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    Structured Review

    Alomone Labs pctx1
    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    2) Product Images from "Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)"

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25386-9

    Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.
    Figure Legend Snippet: Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.

    Techniques Used: Concentration Assay

    Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.
    Figure Legend Snippet: Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.

    Techniques Used: Stable Transfection, Concentration Assay, Inhibition

    PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P
    Figure Legend Snippet: PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P

    Techniques Used: Activation Assay

    Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p
    Figure Legend Snippet: Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p

    Techniques Used: Concentration Assay

    pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).
    Figure Legend Snippet: pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).

    Techniques Used: Inhibition, Stable Transfection, Concentration Assay

    Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.
    Figure Legend Snippet: Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.

    Techniques Used: Concentration Assay

    3) Product Images from "ASIC1a senses lactate uptake to regulate metabolism in neurons"

    Article Title: ASIC1a senses lactate uptake to regulate metabolism in neurons

    Journal: Redox Biology

    doi: 10.1016/j.redox.2022.102253

    ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p
    Figure Legend Snippet: ASIC1a mediates L -lactate-induced increase in mitochondrial respiration and suppresses mitochondrial ROS production . Seahorse analysis (see Materials and Methods) was used to monitor mitochondrial respiration (OCR) with sequential additions of oligomycin (1 μM), FCCP (1 μM) + sodium pyruvate (5 mM), and a mix of rotenone/antimycin A (Ro/AA, 0.5 μM each), as indicated by the arrowheads, in media that contained or not D-, or L-lactate (5 mM); (A) Representative OCR plots of WT neurons in regular medium that contained no lactate (Ctrl) or the indicated lactate isomer and CIN4; (B) Quantification of maximal respiration as measured in (A) of WT neurons in regular medium (n = 6), and the medium that contained D- (n = 6) or L-lactate (n = 6), or L-lactate plus CIN4 (n = 6); (C) Representative OCR plots of KO neurons in regular medium or medium that contained the indicated lactate isomer and CIN4; (D) Quantification of maximal respiration as measured in (C) of KO in regular medium (Ctrl, n = 6) and media that contained the indicated D-lactate (n = 6), L-lactate (n-6), and L-lactate + CIN4 (n = 6); (E) Representative OCR plots of WT and KO neurons in L -lactate containing medium; (F) Quantification of maximal respiration as measured in (E) of WT (n = 6) and KO (n = 6) neurons; (G) Representative OCR plots of WT and KO neurons in L-lactate/CIN4 containing medium (n = 6); (H) Quantification of maximal respiration as measured in (G) of WT and KO neurons in L -lactate/CIN4 containing medium (n = 6 for each); (I) Representative RoGFP fluorescence traces for redox changes in response to L-lactate, H 2 O 2 and DTT added in the Ringer's solution in WT neurons untreated and treated with PcTX1; (J) Quantification of R/R 0 (480/405) at 500s after the addition of L-lactate (100s) as in (I) for WT neurons untreated (n = 9) and treated with PcTX1 (n = 7); (K) Schematic presentation of suggested pathway linking L-lactate to ASIC1a. All summary graph data represent mean ± SD, *p

    Techniques Used: Fluorescence

    Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p
    Figure Legend Snippet: Racemic lactate triggers ASIC1a dependent [Ca 2+ ] c and [Ca 2+ ] m signals. (A) Illustration of the figure hypothesis; (B) Representative Fura-2 ratio traces for [Ca 2+ ] c changes monitored in WT primary cultured hippocampal neurons (DIV 10–15) without and with pretreatment of the selective ASIC1a inhibitor PcTx1 (20 nM, 120 s). Neurons were loaded with Fura-2AM (1 μM) and initially superfused with Ringer's solution at pH 7.4. Then, the superfusion was switched to Ringer's solution of pH 7.4 with an addition of DL-lactate (5 mM) as indicated by the arrowhead; (C) Quantification of the number of cytosolic (cyto) Ca 2+ peaks (transients) per 180-s time period measured as in (B) for WT neurons untreated (n = 39) and treated (n = 77); Box and whiskers plot show maximal and minimal values and all data points; (D) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in Rhod-2 AM (1 μM)-loaded WT neurons untreated and treated with PcTX1. Superfusion was switched to pH 7.4 Ringer's solution with DL-lactate; (E) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (D) for WT neurons untreated (n = 11) and treated with PcTX1 (n = 65); (F) Representative Rhod-2 fluorescence traces for [Ca 2+ ] m changes in response to direct puffing of the pH 7.4-DL- lactate solution in primary cultured WT and KO cortical neurons; (G) Quantification of peak [Ca 2+ ] m based on F/F 0 during the maximum phases as in (F) for WT (n = 6) and KO cortical neurons (n = 5); All summary data represent mean ± SD, ****p

    Techniques Used: Cell Culture, Fluorescence

    4) Product Images from "Structure and analysis of nanobody binding to the human ASIC1a ion channel"

    Article Title: Structure and analysis of nanobody binding to the human ASIC1a ion channel

    Journal: eLife

    doi: 10.7554/eLife.67115

    Effects of Nb.C1 on MitTx and PcTx1 binding to hASIC1a. ( A ) Representative currents of an oocyte expressing hASIC1a activated with pH 6.0 followed by a second activation with 50 nM MitTx at pH 7.4. ( B ) Same experiment after pre-incubation of the oocyte with 50 nM Nb.C1 for 15 min. ( C ) Summary of the peak currents from pH 6.0 and MitTx activations. In this and all traces, the conditioning pH is 7.4. The bars represent the mean±SD of currents, n=8 Nb control and n=6 Nb.C1. Asterisks indicate statistical significance in t-test, p
    Figure Legend Snippet: Effects of Nb.C1 on MitTx and PcTx1 binding to hASIC1a. ( A ) Representative currents of an oocyte expressing hASIC1a activated with pH 6.0 followed by a second activation with 50 nM MitTx at pH 7.4. ( B ) Same experiment after pre-incubation of the oocyte with 50 nM Nb.C1 for 15 min. ( C ) Summary of the peak currents from pH 6.0 and MitTx activations. In this and all traces, the conditioning pH is 7.4. The bars represent the mean±SD of currents, n=8 Nb control and n=6 Nb.C1. Asterisks indicate statistical significance in t-test, p

    Techniques Used: Binding Assay, Expressing, Activation Assay, Incubation

    Structural comparison of hASIC1a-Nb.C1 complex to toxin-bound ASICs. Two side, top and bottom views of superimposed structures of hASIC1a-NbC1 complex (red) with ( A ) MitTx-bound to chicken ASIC1 (4ntw) in open conformation (orange). In side views, the threefold axis of the channel is indicated by a dashed vertical line; in top and bottom views it is indicated by dotted triangles. ( B ) PcTx1-bound chicken ASIC1 (3s3x) (gray). ( C ) Mambalgin-1-bound human ASIC1 (7ctf) (blue). Only one subunit is shown for simplicity. Surface clashes are indicated by dashed rectangles. Nb.C1, MitTx- α, MitTx- β, PcTx1, Mambalgin-1 are shown as red, orange, light-orange, light-purple, marine respectively.
    Figure Legend Snippet: Structural comparison of hASIC1a-Nb.C1 complex to toxin-bound ASICs. Two side, top and bottom views of superimposed structures of hASIC1a-NbC1 complex (red) with ( A ) MitTx-bound to chicken ASIC1 (4ntw) in open conformation (orange). In side views, the threefold axis of the channel is indicated by a dashed vertical line; in top and bottom views it is indicated by dotted triangles. ( B ) PcTx1-bound chicken ASIC1 (3s3x) (gray). ( C ) Mambalgin-1-bound human ASIC1 (7ctf) (blue). Only one subunit is shown for simplicity. Surface clashes are indicated by dashed rectangles. Nb.C1, MitTx- α, MitTx- β, PcTx1, Mambalgin-1 are shown as red, orange, light-orange, light-purple, marine respectively.

    Techniques Used:

    5) Product Images from "Histidine Residues Are Responsible for Bidirectional Effects of Zinc on Acid-Sensing Ion Channel 1a/3 Heteromeric Channels"

    Article Title: Histidine Residues Are Responsible for Bidirectional Effects of Zinc on Acid-Sensing Ion Channel 1a/3 Heteromeric Channels

    Journal: Biomolecules

    doi: 10.3390/biom10091264

    Co-overexpression of 1:2, but not 2:1 ratio of ASIC1a and ASIC3 cDNA revealed a profound response to zinc. ( A ) Activation of heteromeric ASIC1a/3 channels by fast perfusion for a drop in pH from 7.4 to 6.5 on CHO cell expressing both ASIC1a and ASIC3 subunits. The perfusion time for low pH value (e.g., 6.5) is 7 s; ( B ) Representative traces show that PcTx1 (10 nM) and APETx2 (100 nM) have no effects on the heteromeric ASIC1a/3 currents using a 1:2 ratio of ASIC1a and ASIC3, n = 5; ( C ) Representative traces show that PcTx1 (10 nM) and APETx2 (100 nM) also have no effects on the heteromeric ASIC1a/3 currents using a 2:1 ratio of ASIC1a and ASIC3, n = 5; ( D ) Co-application and pretreatment with zinc at 50 µM significantly potentiated the currents of heteromeric ASIC1a/3 using a 1:2 ratio of ASIC1a and ASIC3 (the same cell as Figure 2 B), n = 5; ( E ) Co-application and pretreatment with zinc at 50 µM had no effects on the currents of heteromeric ASIC1a/3 using a 2:1 ratio of ASIC1a and ASIC3 (the same cell as Figure 2 C), n = 5. Dashed black line represents pretreatment with zinc in pH 7.4 extracellular solution (2 min duration).
    Figure Legend Snippet: Co-overexpression of 1:2, but not 2:1 ratio of ASIC1a and ASIC3 cDNA revealed a profound response to zinc. ( A ) Activation of heteromeric ASIC1a/3 channels by fast perfusion for a drop in pH from 7.4 to 6.5 on CHO cell expressing both ASIC1a and ASIC3 subunits. The perfusion time for low pH value (e.g., 6.5) is 7 s; ( B ) Representative traces show that PcTx1 (10 nM) and APETx2 (100 nM) have no effects on the heteromeric ASIC1a/3 currents using a 1:2 ratio of ASIC1a and ASIC3, n = 5; ( C ) Representative traces show that PcTx1 (10 nM) and APETx2 (100 nM) also have no effects on the heteromeric ASIC1a/3 currents using a 2:1 ratio of ASIC1a and ASIC3, n = 5; ( D ) Co-application and pretreatment with zinc at 50 µM significantly potentiated the currents of heteromeric ASIC1a/3 using a 1:2 ratio of ASIC1a and ASIC3 (the same cell as Figure 2 B), n = 5; ( E ) Co-application and pretreatment with zinc at 50 µM had no effects on the currents of heteromeric ASIC1a/3 using a 2:1 ratio of ASIC1a and ASIC3 (the same cell as Figure 2 C), n = 5. Dashed black line represents pretreatment with zinc in pH 7.4 extracellular solution (2 min duration).

    Techniques Used: Over Expression, Activation Assay, Expressing

    6) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    7) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    8) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    9) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    10) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    11) Product Images from "Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)"

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-25386-9

    Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.
    Figure Legend Snippet: Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.

    Techniques Used: Concentration Assay, Size-exclusion Chromatography

    Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.
    Figure Legend Snippet: Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.

    Techniques Used: Stable Transfection, Concentration Assay, Inhibition

    PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P
    Figure Legend Snippet: PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P

    Techniques Used: Activation Assay

    Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p
    Figure Legend Snippet: Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p

    Techniques Used: Concentration Assay, Size-exclusion Chromatography

    pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).
    Figure Legend Snippet: pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).

    Techniques Used: Inhibition, Stable Transfection, Concentration Assay

    Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.
    Figure Legend Snippet: Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.

    Techniques Used: Concentration Assay

    12) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    13) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    14) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    15) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    16) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    17) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    18) Product Images from "ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons"

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    Journal: Journal of neurochemistry

    doi: 10.1111/jnc.14971

    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Techniques Used: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p
    Figure Legend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Techniques Used: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Figure Legend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Techniques Used: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

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    Alomone Labs pctx1
    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor <t>PcTX1</t> (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p
    Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs psalmotoxin 1 pctx1
    Effect of amiloride, mambalgin-1, APETx2 and <t>PcTx1</t> on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.
    Psalmotoxin 1 Pctx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Journal: Journal of neurochemistry

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    doi: 10.1111/jnc.14971

    Figure Lengend Snippet: Activation of ASIC1a at pH 7.0 triggers cytosolic and mitochondrial Na + signals (A) Representative fluorescence traces of cytosolic Na + concentration ([Na + ] c ) changes monitored in WT primary cultured cortical neurons (DIV 10-15) without and with pretreatment of the selective ASIC1a inhibitor PcTX1 (20 nM, 120 s). Neurons were loaded with Asante NaTRIUM Green-2 (1 μM) and initially superfused with Ringer’s solution at pH 7.4. Then, the superfusion was switched to Ringer’s solution of pH 7.0 as indicated. (B) Quantification of [Na + ] c change rates measured as in (A) for WT neurons untreated (n=3) and treated with PcTX1 (n=6). (C) Immunoblot analysis of ASIC1a in WT and ASIC1a KO neurons. GAPDH was used as a loading control, representative of n = 3 independent experiments. (D) Representative fluorescence traces of [Na + ] c changes in WT and ASIC1a KO neurons loaded with Asante NaTRIUM Green in response to pH 7.0 Ringer’s solution applied through direct puffing. (E) Quantification of rates of [Na + ] c changes during the early phases as in (D) for WT (n=3) and ASIC1a KO (n=3) neurons. ). (G) Representative [Na + ]c changes in response to superfusion of the pH 7.0 solution in HEK293-T cells transfected with the empty vector and overexpressing ASIC1a without and with PcTX1 pretreatment as in (A). (H) Quantification of rates of [Na + ] c changes as in (E) for pcDNA-transfected (n=3), ASIC1a overexpressing HEK 293-T cells untreated (n=3) and treated (n=3) with PcTX1. (I) Representative images (x10) of neurons that were loaded with Asante NaTRIUM Green-2 before and after before pH change in the presence of Na + . The scale bars represent 100 μm. (J) Representative fluorescence traces for mitochondrial Na + concentration ([Na + ] m ) changes in response to superfusion of the pH 7.0 solution in WT neurons loaded with CoroNa Red (1 μM) untreated and pretreated with PcTX1. (K) Quantification of rates of [Na + ] m increases as in (I) for WT neurons untreated (n=3) and treated with PcTX1 (n = 4). (L) Resresentative images of neurons (x20) that were loaded with CoroNa Red before and after pH shift in the presence of Na + . The scale bars represent 50 μm. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Article Snippet: With the pHe shifted from 7.4 to 7.0, a rise in mitochondrial Na+ concentration was observed, which was partially blocked by PcTX1, indicating that the cytosolic Na+ rise mediated by ASIC1a is propagated into the mitochondria.

    Techniques: Activation Assay, Fluorescence, Concentration Assay, Cell Culture, Transfection, Plasmid Preparation

    Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Journal: Journal of neurochemistry

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    doi: 10.1111/jnc.14971

    Figure Lengend Snippet: Physiological pH shift from 7.4 to 7.0 triggers an ASIC1a-dependent change in neuronal mitochondrial metabolic activity. ) were used to monitor basal, maximal and ATP-coupled respiration at pH ~6.5, after addition of Oligomycin (Oligo. 1 μM), FCCP (1 μM) + sodium pyruvate (5 mM) (FCCP) and antimycin A (4 μM) as indicated by the arrowheads. The low pH e environment (pH~6.5) was induced by evoked glycolysis in medium that contained low glucose and culturing without CO 2 . (A) Extracellular acidification rate (ECAR) levels showing the exact pH levels of the extracellular medium during 120 minutes of the experiment. (B) Representative Oxygen Consumption Rate (OCR) traces ± SEM measured in WT neurons with or without PcTX1 treatment. (C) Quantification of basal, maximal, ATP-coupled and non-mitochondrial respiration as measured in (A) for untreated (n=4-5) and PcTx1-pretreated (n=4-5) WT neurons. (D) Representative traces of OCR ± SEM of WT and ASIC1a KO neurons. (E) Quantification of OCR parameters as determined in (D) for WT (n=3) ASIC1 KO (n=3) neurons. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant, *p

    Article Snippet: With the pHe shifted from 7.4 to 7.0, a rise in mitochondrial Na+ concentration was observed, which was partially blocked by PcTX1, indicating that the cytosolic Na+ rise mediated by ASIC1a is propagated into the mitochondria.

    Techniques: Activity Assay

    Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Journal: Journal of neurochemistry

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    doi: 10.1111/jnc.14971

    Figure Lengend Snippet: Mitochondrial ASIC1a plays a role in mitochondrial Na + signaling (A) Representative [Na + ] m ). Cells were loaded with CoroNa Red (1 μM) and then untreated or treated with PcTX1 before the pH 7.0 solution was applied through superfusion. (B) Quantification of the rate of [Na + ] m increase measured in (A) for cells untreated (n=3) and treated (n=3) with PcTX1. (C) Representative fluorescence traces of [Na + ] m changes in response to superfusion of the pH 7.0 solution of untreated and PcTX1 pre-treated digitonin-permeabilized WT neurons. (D) Quantification of the rate of [Na + ] m increase determined in (C) for WT neurons untreated (n = 3) and pretreated (n=3) with PcTX1. All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures, ns non-significant **p

    Article Snippet: With the pHe shifted from 7.4 to 7.0, a rise in mitochondrial Na+ concentration was observed, which was partially blocked by PcTX1, indicating that the cytosolic Na+ rise mediated by ASIC1a is propagated into the mitochondria.

    Techniques: Fluorescence

    Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Journal: Journal of neurochemistry

    Article Title: ASIC1a Channels Regulate Mitochondrial Ion Signaling and Energy Homeostasis in Neurons

    doi: 10.1111/jnc.14971

    Figure Lengend Snippet: Physiological pH shift from 7.4 to 7.0 triggers a cytosolic Ca 2+ response that is propagated to mitochondria A) Representative fluorescence ratio traces of pH-dependent cytosolic Ca 2+ concentration ([Ca 2+ ] c ) changes monitored in untreated and PcTX1-pretreated WT neurons. Neurons were loaded with Fura-2-AM (1 μM) and pH change was carried out through superfusion. (B) Quantification of [Ca 2+ ] c increase as measured in (A) for untreated (n=3) and PcTX1-pretreated (n=3) neurons. (C) Representative fluorescence ratio traces of [Ca 2+ ] c changes induced by puffing with the pH 7.0 solution in WT and ASIC1a KO neurons loaded with Fura-2-AM. (D) Quantification of [Ca 2+ ] c increase as determined in (C) for WT (n=3) and ASIC1a KO (n=3) neurons. (E) Reresentative images of neurons (x10) that were loaded with Fura-2-AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 100 μm. (F) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2 AM (1 μM) loaded WT neurons untreated and pretreated with PcTX1. (G) Quantification of [Ca 2+ ] m changes as measured in (E) for untreated (n=7) and PcTX1-pretreated (n=7) neurons. (H) Representative fluorescence traces of [Ca 2+ ] m changes induced by puffing the pH 7.0 solution in WT and ASIC1a KO neurons. (I) Quantification of [Ca 2+ ] m changes determined as in (G) for WT (n=3) and ASIC1a KO (n=4) neurons. (J) Representative images of neurons (x20) that were loaded with Rhod-2 AM before and after pH shift in the presence of Ca 2+ . The scale bars represent 50 μm. (K) Representative fluorescence traces of pH-dependent [Ca 2+ ] m changes induced through superfusion in HEK293-T cells that overexpressed ASIC1a or were transfected with the empty vector (pcDNA). (L) Quantification [Ca 2+ ] m changes determined as in (I) for HEK293-T cells that overexpressed ASIC1a (n=3) and were transfected with pcDNA vector (n=3). (M) Representative pH-dependent mitochondrial Ca 2+ concentration ([Ca 2+ ] m ) changes induced by superfusion and monitored by fluorescence in Rhod-2AM (1 μM) loaded WT neurons pretreated with Nifedipin and PcTX1 separately. (N) Quantification of [Ca 2+ ] m changes as measured in (K) for untreated (n=3), Nifedipin (n=3) and PcTX1-pretreated (n=3) neurons All bar graph data represent mean ± SEM, with n representing the number of independent cell cultures. *p

    Article Snippet: With the pHe shifted from 7.4 to 7.0, a rise in mitochondrial Na+ concentration was observed, which was partially blocked by PcTX1, indicating that the cytosolic Na+ rise mediated by ASIC1a is propagated into the mitochondria.

    Techniques: Fluorescence, Concentration Assay, Transfection, Plasmid Preparation

    Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: Potentiation of ASIC1a/2a by PcTx1 in rat cortical neurons. ( a ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of PcTx1. (b and c) Time courses of PcTx1 modulation for the same neuron as in ( a ). The dotted lines at the bottom represent the baseline. Note the dip in membrane potential to below that in the presence of 10 nM PcTx1 upon initial wash of 10 nM ( b ) and 100 nM ( c ) PcTx1. ( d ) Averages of pH6.8-induced depolarization from multiple neurons (n = 8, 4, 8 and 8 for 0, 1, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. ( e ) Traces of pH6.8-induced current from a single neuron in the absence and presence of PcTx1. ( f ) Time course of PcTx1 modulation from the same neuron as in ( e ). The dotted line at the top represents the baseline. Note the lower current amplitude than that in the presence of 10 nM PcTx1 upon first removal of 100 nM PcTx1. ( g ) Averages of pH6.8-induced currents from multiple neurons (n = 10 each for 0, 10 and 100 nM PcTx1, respectively). The horizontal dashed line represents the value in the presence of 10 nM PcTx1. PcTx1 was applied in the order of increasing concentration and the wash data were recorded upon wash-off of 100 nM PcTx1. For all the experiments in Fig. 4, the conditioning pH was 7.4 and the test pH of 6.8 was applied for 2–3 sec before returning to pH7.4. This pH application protocol was repeated once every 60 sec.

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Concentration Assay

    Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: Potentiation by PcTx1 of ASIC1a/2a stably expressed in CHO cells. ( a ) Traces of pH6.8- and pH6.0-induced membrane depolarization from a single cell in the absence and presence of 0.3–300 nM PcTx1. PcTx1 was continuously applied in the order of increasing concentration till reaching steady state at each concentration. Note that the decrease in pH6.8-induced depolarization in the presence of 0.3 nM and 1 nM PcTx1 was reversed by 10–300 nM PcTx1. ( b ) Current traces from a single cell showing inhibition by 1 nM PcTx1 and potentiation by 10–300 nM PcTx1 of pH6.8-induced current responses. ( c ) Average % change in the amplitude of pH6.8-induced depolarization (solid squares; n = 7) and peak current (open circles; n = 6) as a function of PcTx1 concentration. Data were normalized to the value at 300 nM PcTx1 for each cell before averaging. Only data between 1 nM and 300 nM PcTx1 were used for curve fitting (dashed and dotted lines). IC 50 values from the fits are 56.1 nM (depolarization) and 123.9 nM (current), respectively. The conditioning pH was 7.4 for all the experiments shown in Fig. 2.

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Stable Transfection, Concentration Assay, Inhibition

    PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: PcTx1 shifted pH activation of ASIC1a/2a toward lower proton concentrations in CHO cells. ( a ) Current traces from a single cell showing activation of ASIC1a/2a at various test pHs (pH6.8–5.7) in the absence of PcTx1. ( b ) Current traces from the same cell as in ( a ) showing activation of ASIC1a/2a at the same test pHs in the presence of 100 nM PcTx1. ( c ) Average current responses as a function of test pH in the absence (solid squares) and presence (open circles) of 100 nM PcTx1. Current amplitudes were normalized to that at pH5.7 for each cell in the absence (n = 6) and presence (n = 4) of PcTx1, respectively. The solid and dashed lines are best fits to the data in the absence (pH 50 = 6.19) and presence (pH 50 = 6.31) of 100 nM PcTx1, respectively. PcTx1 significantly (P

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Activation Assay

    Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: Potentiation of ASIC1a/2a by low concentration PcTx1 in mouse cortical neurons. ( a ) Correlation between pH6.8-induced current and membrane depolarization in wild-type (solid squares; n = 17) and ASIC1 −/− (open circles; n = 16) neurons. Each symbol represents one neuron. ( b ) Traces of pH6.8-induced membrane depolarization from a single neuron in the absence and presence of 10 nM PcTx1. Inset: Time course of the effect of 10 nM PcTx1 and wash. The dotted line at the bottom represents the baseline. ( c ) Time courses of pH6.8-induced responses upon removal of 10 nM PcTx1. Wash began at t = 0 sec as indicated by the green arrow. Open squares: current (n = 4); solid squares: membrane potential (n = 4). Responses were normalized to the control values for each cell just before application of 10 nM PcTx1. Note the initial lower responses (at t = 60 sec) than those in the presence of 10 nM PcTx1 (at t ≤ 0 sec). ( d ) Summary of pH6.8-induced membrane depolarization in wild-type and ASIC1 −/− neurons. Values for wild-type neurons during and upon initial wash of 10 nM PcTx1 were both significantly higher than that from ASIC1 −/− neurons (p

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Concentration Assay

    pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: pH dependence of PcTx1 inhibition of ASIC1a/2a stably expressed in CHO cells. ( a ) Current traces from a single cell showing that PcTx1 (1 nM) had no effect on pH5.7-induced currents at the conditioning pH of 7.4. ( b ) Current traces from a single cell showing the dependence of pH6.0-induced current response on the conditioning pH as indicated. ( c ) Average (n = 5) of pH6.0-induced current amplitude (normalized to the value at conditioning pH7.1 for each cell before averaging) as a function of conditioning pH. pH 50 = 6.94 from the best fit (dashed line). The holding potential was −80 mV. ( d ) Current traces from a single cell showing the inhibition of pH6.0-induced current at various PcTx1 concentrations (1–300 nM). The conditioning pH was 7.0. ( e ) Time course of PcTx1 modulation from a single cell. The dotted line at the top represents the current baseline. ( f ) Average % inhibition (n = 4–6) of pH6.0-induced current as a function of PcTx1 concentration. IC 50 = 2.9 nM and the maximal inhibition was 75.7% from the best fit (dashed line).

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Inhibition, Stable Transfection, Concentration Assay

    Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.

    Journal: Scientific Reports

    Article Title: Dual actions of Psalmotoxin at ASIC1a and ASIC2a heteromeric channels (ASIC1a/2a)

    doi: 10.1038/s41598-018-25386-9

    Figure Lengend Snippet: Potentiation of ASIC1a/2a by high concentration PcTx1 in mouse cortical neurons. ( a ) Traces of pH6.8-induced current from a single wild-type neuron in control buffer and in the presence of 10 nM PcTx1, 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( b ) Traces of pH6.8- or pH4.5-induced current from a single ASIC1 −/− neuron in control buffer (for both pH6.8 and pH4.5) and (for pH6.8 only) in the presence of 100 nM PcTx1, 100 nM PcTx1 + 300 μM Zn and 100 nM PcTx1 + 300 μM Zn + 500 μM amiloride. ( c ) Summary of pH6.8, pH6.0 or pH4.5-induced currents under conditions indicated for wild-type and ASIC1 −/− neurons. Numbers in the parentheses indicate the number of neurons tested under each indicated condition. The conditioning pH was 7.4 for all the experiments in Fig. 6.

    Article Snippet: PcTx1 was purchased from Alomone Labs (Israel).

    Techniques: Concentration Assay

    Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Journal: Frontiers in Neuroscience

    Article Title: Involvement of Acid-Sensing Ion Channel 1b in the Development of Acid-Induced Chronic Muscle Pain

    doi: 10.3389/fnins.2019.01247

    Figure Lengend Snippet: Effect of amiloride, mambalgin-1, APETx2 and PcTx1 on ASIC1b-expressing muscle afferent DRG neurons. (A) Whole-cell patch clamp recording on an ASIC1b-expressing DRG neuron projecting to gastrocnemius muscle labeled by fluorogold. (B) Mambalgin-1 (MB-1) (1 μM) inhibited acid (pH 5.0)-induced currents in 13 of 14 ASIC1b-expressing muscle afferent DRG neurons. (C) APETx2 (1 μM) inhibited acid (pH 5.0)-induced currents in 6 of 13 ASIC1b-expressing muscle afferent DRG neurons. (D) PcTx1 (100 nM) inhibited acid (pH 5.0)-induced currents in 5 of 11 ASIC1b-expressing muscle afferent DRG neurons.

    Article Snippet: Mambalgin-1, APETx2, and psalmotoxin 1 (PcTx1) were purchased from Alomone Labs and prepared by autoclaved water in stock solutions of 200 μM, 1 mM, and 200 μM respectively.

    Techniques: Expressing, Patch Clamp, Labeling

    ASIC1a channels presynaptically control transmitter release in motor nerve terminals from female mice. A : average spontaneous miniature end-plate potential (MEPP) frequencies obtained from female ( left ) and male ( right ) wild-type (filled black circles), wild type + psalmotoxin-1 (10 nM, open black circles), and ASIC1a −/− (filled gray circles) levator auris longus muscle motor nerve terminals (MNTs). In female mice, wild-type frequencies were significantly lower compared with both wild type + psalmotoxin-1 (10 nM) and ASIC1a −/− [1-way ANOVA, F (2,61) = 23.1; Bonferroni post hoc test, P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice

    doi: 10.1152/ajpcell.00301.2013

    Figure Lengend Snippet: ASIC1a channels presynaptically control transmitter release in motor nerve terminals from female mice. A : average spontaneous miniature end-plate potential (MEPP) frequencies obtained from female ( left ) and male ( right ) wild-type (filled black circles), wild type + psalmotoxin-1 (10 nM, open black circles), and ASIC1a −/− (filled gray circles) levator auris longus muscle motor nerve terminals (MNTs). In female mice, wild-type frequencies were significantly lower compared with both wild type + psalmotoxin-1 (10 nM) and ASIC1a −/− [1-way ANOVA, F (2,61) = 23.1; Bonferroni post hoc test, P

    Article Snippet: Toxin and chemicals.Alpha-bungarotoxin and all salts of analytical grade were purchased from Sigma (St. Louis, MO), and μ-conotoxin GIIIB and psalmotoxin-1 were purchased from Alomone Labs (Jerusamen, Israel).

    Techniques: Mouse Assay

    ASIC1a channels reduced neuromuscular transmission during 75 Hz/5 s stimulation train in motor nerve terminals of female mice. A : representative intracellular recordings of EPP are shown during nerve stimulation at 75 Hz (5 s long, 375 stimuli) using 0.8 mM HEPES/2 mM [Ca 2+ ]/1 mM [Mg 2+ ] + 10 μM μ-conotoxin GIIIB saline solution. EPP facilitate during the first stimuli and then undergo depression until a steady state was reached (top trace, wild type). Interestingly, EPP from ASIC1a −/− female mice (mid trace) and female wild type + psalmotoxin 1 (10 nM; bottom trace) presented greater initial facilitation compared with those from wild-type female mice (top trace). B : average EPP amplitude ratio [( n th stimuli amplitude/1st stimulus amplitude) × 100] of the initial 50 stimuli shown in A for levator auris longus MNTs from wild type (filled circles, n = 8 MNTs), ASIC1a −/− (open circles, n = 6 MNTs), and wild type + psalmotoxin-1 (10 nM; filled triangles, n = 5 MNTs). Wild-type ratios were significantly smaller than both wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ones [one-way ANOVA, F (2,87) = 4.3; wild type vs. ASIC1a −/− , wild type + psalmotoxin-1 (10 nM), Bonferroni post hoc test, P = 0.016]. Wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ratios were not significantly different (Bonferroni post hoc test, P = 1.0). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Acid-sensing ion channels 1a (ASIC1a) inhibit neuromuscular transmission in female mice

    doi: 10.1152/ajpcell.00301.2013

    Figure Lengend Snippet: ASIC1a channels reduced neuromuscular transmission during 75 Hz/5 s stimulation train in motor nerve terminals of female mice. A : representative intracellular recordings of EPP are shown during nerve stimulation at 75 Hz (5 s long, 375 stimuli) using 0.8 mM HEPES/2 mM [Ca 2+ ]/1 mM [Mg 2+ ] + 10 μM μ-conotoxin GIIIB saline solution. EPP facilitate during the first stimuli and then undergo depression until a steady state was reached (top trace, wild type). Interestingly, EPP from ASIC1a −/− female mice (mid trace) and female wild type + psalmotoxin 1 (10 nM; bottom trace) presented greater initial facilitation compared with those from wild-type female mice (top trace). B : average EPP amplitude ratio [( n th stimuli amplitude/1st stimulus amplitude) × 100] of the initial 50 stimuli shown in A for levator auris longus MNTs from wild type (filled circles, n = 8 MNTs), ASIC1a −/− (open circles, n = 6 MNTs), and wild type + psalmotoxin-1 (10 nM; filled triangles, n = 5 MNTs). Wild-type ratios were significantly smaller than both wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ones [one-way ANOVA, F (2,87) = 4.3; wild type vs. ASIC1a −/− , wild type + psalmotoxin-1 (10 nM), Bonferroni post hoc test, P = 0.016]. Wild-type + psalmotoxin-1 (10 nM) and ASIC1a −/− ratios were not significantly different (Bonferroni post hoc test, P = 1.0). * P

    Article Snippet: Toxin and chemicals.Alpha-bungarotoxin and all salts of analytical grade were purchased from Sigma (St. Louis, MO), and μ-conotoxin GIIIB and psalmotoxin-1 were purchased from Alomone Labs (Jerusamen, Israel).

    Techniques: Transmission Assay, Mouse Assay