pctx1  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Alomone Labs pctx1
    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 <t>PcTx1</t> (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 < 0.0001.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) 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

    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 < 0.0001.
    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 < 0.0001.

    Techniques Used: Cell Culture, Fluorescence

    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 < 0.05, **p < 0.01, ***p < 0.001.
    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 < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Fluorescence

    synthetic pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs synthetic pctx1
    ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. <t>PcTx1</t> (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .
    Synthetic 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
    https://www.bioz.com/result/synthetic pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition"

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    Journal: eLife

    doi: 10.7554/eLife.73384

    ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .
    Figure Legend Snippet: ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .

    Techniques Used: Labeling, Activation Assay, Concentration Assay, Fluorescence

    ( A ) Representative trace of a VCF recording of K105C* showing application of PcTx1 (300 nM) washed off for 3 min with alternating pHs (20 s pH 7.4, 60 s pH 8.4, 40 s pH 7.4, 60 s pH 8.4) before switching to pH 7.4 again. ( B ) Comparison of the fluorescence of K105C* after a 3 min washout of 300 nM PcTx1 using pH 7.4, 8.4, or a mix of the two as shown in the protocol in ( A ) and . ( C ) Same as in ( A ) but for V80C* and running buffer 7.7 instead of 7.4. ( D ) Same as in ( B ) but for V80C* and base on the protocol shown in ( C ) and . All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( B ) and ( D ) are presented as mean ± 95 CI, ordinary analysis of variance (ANOVA).
    Figure Legend Snippet: ( A ) Representative trace of a VCF recording of K105C* showing application of PcTx1 (300 nM) washed off for 3 min with alternating pHs (20 s pH 7.4, 60 s pH 8.4, 40 s pH 7.4, 60 s pH 8.4) before switching to pH 7.4 again. ( B ) Comparison of the fluorescence of K105C* after a 3 min washout of 300 nM PcTx1 using pH 7.4, 8.4, or a mix of the two as shown in the protocol in ( A ) and . ( C ) Same as in ( A ) but for V80C* and running buffer 7.7 instead of 7.4. ( D ) Same as in ( B ) but for V80C* and base on the protocol shown in ( C ) and . All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( B ) and ( D ) are presented as mean ± 95 CI, ordinary analysis of variance (ANOVA).

    Techniques Used: Fluorescence

    ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .
    Figure Legend Snippet: ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .

    Techniques Used: Binding Assay, Activation Assay, Functional Assay, Fluorescence

    ( A ) Representative VCF trace of K105C* showing application of PcTx1 (300 nM) washout for 3 min before pH 5.5 stimulus. Corresponding binding modes are indicated below. ( B ) Comparison of the final pH 5.5-induced current (I) and fluorescence (ΔF) at pH 7.4 in recordings shown in , where channels undergo three 1 min washouts at pH 7.4, each followed by pH 5.5 stimulus (left) or the protocol shown in ( A ) with a single 3 min washout (right). The final pH 5.5-induced current was normalized to the one at the beginning of the recording, the fluorescence was analyzed right before the final pH 5.5 activation and normalized to the deflection induced by PcTx1. ( C ) Representative VCF trace of K105C* showing pH 7.0 stimuli at various states of the recording. Corresponding binding modes are indicated below. ( D ) Representative trace of a VCF recording of K105C* depicting 30 s pre-conditioning with big dynorphin (BigDyn) (1 μM) and subsequent 30 s PcTx1 (300 nM) application and pH 5.5 activation. ( E ) Quantitative comparison of the fluorescence change induced by 300 nM PcTx1 at pH 7.4 in the apo state (control) and after BigDyn (1 μΜ) application, normalized to the signal induced by pH 5.5. Data in ( B ) and ( E ) are presented as mean ± 95CI.
    Figure Legend Snippet: ( A ) Representative VCF trace of K105C* showing application of PcTx1 (300 nM) washout for 3 min before pH 5.5 stimulus. Corresponding binding modes are indicated below. ( B ) Comparison of the final pH 5.5-induced current (I) and fluorescence (ΔF) at pH 7.4 in recordings shown in , where channels undergo three 1 min washouts at pH 7.4, each followed by pH 5.5 stimulus (left) or the protocol shown in ( A ) with a single 3 min washout (right). The final pH 5.5-induced current was normalized to the one at the beginning of the recording, the fluorescence was analyzed right before the final pH 5.5 activation and normalized to the deflection induced by PcTx1. ( C ) Representative VCF trace of K105C* showing pH 7.0 stimuli at various states of the recording. Corresponding binding modes are indicated below. ( D ) Representative trace of a VCF recording of K105C* depicting 30 s pre-conditioning with big dynorphin (BigDyn) (1 μM) and subsequent 30 s PcTx1 (300 nM) application and pH 5.5 activation. ( E ) Quantitative comparison of the fluorescence change induced by 300 nM PcTx1 at pH 7.4 in the apo state (control) and after BigDyn (1 μΜ) application, normalized to the signal induced by pH 5.5. Data in ( B ) and ( E ) are presented as mean ± 95CI.

    Techniques Used: Binding Assay, Fluorescence, Activation Assay

    ( A ) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two, or all three subunits. ( B ) Representative two-electrode voltage-clamp (TEVC) trace of activation ( C ) Activation curve from recordings shown in ( B ) for the four different concatemeric constructs (n = 7–13). ( D ) Representative TEVC trace of steady-state desensitization (SSD). ( E ) SSD profiles from recordings shown in ( D ) (n = 4–11). ( F ) Representative TEVC trace of concentration-dependent PcTx1 inhibition at pH 7.4. ( G ) PcTx1 concentration–response curves from data shown in ( F ) (n = 4–11). Scale bars are 4 µA (vertical) and 60 s (horizontal) for (B, D) and 30 s for (F). Data points in ( C, E and G ) represent mean ± 95CI. Figure 4—source data 1. TEVC data from concatemeric mASIC1a of pH-dependent activation, as shown in and . Figure 4—source data 2. TEVC data from concatemeric mASIC1a of SSD, as shown in . Figure 4—source data 3. TEVC data from concatemeric mASIC1a of PcTx1 concentration–response curve, as shown in and .
    Figure Legend Snippet: ( A ) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two, or all three subunits. ( B ) Representative two-electrode voltage-clamp (TEVC) trace of activation ( C ) Activation curve from recordings shown in ( B ) for the four different concatemeric constructs (n = 7–13). ( D ) Representative TEVC trace of steady-state desensitization (SSD). ( E ) SSD profiles from recordings shown in ( D ) (n = 4–11). ( F ) Representative TEVC trace of concentration-dependent PcTx1 inhibition at pH 7.4. ( G ) PcTx1 concentration–response curves from data shown in ( F ) (n = 4–11). Scale bars are 4 µA (vertical) and 60 s (horizontal) for (B, D) and 30 s for (F). Data points in ( C, E and G ) represent mean ± 95CI. Figure 4—source data 1. TEVC data from concatemeric mASIC1a of pH-dependent activation, as shown in and . Figure 4—source data 2. TEVC data from concatemeric mASIC1a of SSD, as shown in . Figure 4—source data 3. TEVC data from concatemeric mASIC1a of PcTx1 concentration–response curve, as shown in and .

    Techniques Used: Construct, Mutagenesis, Activation Assay, Concentration Assay, Inhibition

    ( A ) Activation and steady-state desensitization (SSD) curves for trimeric and concatemeric WT and F350L channels in comparison. ( B ) Activation curves for all concatemeric variants showing that concatemers with the same number of F350L-bearing subunits cluster around similar pH sensitivities. ( C ) Same as in ( B ), but for concentration-dependent PcTx1 inhibition. Data are presented as mean ± 95 CI.
    Figure Legend Snippet: ( A ) Activation and steady-state desensitization (SSD) curves for trimeric and concatemeric WT and F350L channels in comparison. ( B ) Activation curves for all concatemeric variants showing that concatemers with the same number of F350L-bearing subunits cluster around similar pH sensitivities. ( C ) Same as in ( B ), but for concentration-dependent PcTx1 inhibition. Data are presented as mean ± 95 CI.

    Techniques Used: Activation Assay, Concentration Assay, Inhibition

    ( A ) Representative voltage-clamp fluorometry (VCF) trace of 300 nM PcTx1 application to a concatemeric construct labeled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. ( B ) Same as in ( A ) but one subunit carries a F350L mutation. ( C ) Same as in ( A ) but with two subunits carry a F350L mutation. ( D ) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in ( A – C ). Results from non-concatenated channels are indicated for comparison (shown in light gray). ( E ) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light gray). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red vertical). In ( D ) and ( E ), error bars represents 95CI, unpaired Mann–Whitney test to neighboring bar on the left, *p<0.05, **p<0.005, ***p<0.0005. Figure 5—source data 1. VCF data from concatemeric mASIC1a of PcTx1 application and washout as shown in .
    Figure Legend Snippet: ( A ) Representative voltage-clamp fluorometry (VCF) trace of 300 nM PcTx1 application to a concatemeric construct labeled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. ( B ) Same as in ( A ) but one subunit carries a F350L mutation. ( C ) Same as in ( A ) but with two subunits carry a F350L mutation. ( D ) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in ( A – C ). Results from non-concatenated channels are indicated for comparison (shown in light gray). ( E ) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light gray). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red vertical). In ( D ) and ( E ), error bars represents 95CI, unpaired Mann–Whitney test to neighboring bar on the left, *p<0.05, **p<0.005, ***p<0.0005. Figure 5—source data 1. VCF data from concatemeric mASIC1a of PcTx1 application and washout as shown in .

    Techniques Used: Construct, Labeling, Mutagenesis, Fluorescence, MANN-WHITNEY

    Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.
    Figure Legend Snippet: Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

    Techniques Used: Binding Assay, Mutagenesis

    pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Alomone Labs pctx1
    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 <t>PcTx1</t> (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 < 0.0001.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) 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

    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 < 0.0001.
    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 < 0.0001.

    Techniques Used: Cell Culture, Fluorescence

    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 < 0.05, **p < 0.01, ***p < 0.001.
    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 < 0.05, **p < 0.01, ***p < 0.001.

    Techniques Used: Fluorescence

    modulation by pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs modulation by pctx1
    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for <t>PcTx1</t> modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.
    Modulation By 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
    https://www.bioz.com/result/modulation by pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    modulation by pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology"

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001321

    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.
    Figure Legend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.

    Techniques Used: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY, Standard Deviation

    (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.
    Figure Legend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.

    Techniques Used: Expressing, Incubation, Purification, Western Blot, Mutagenesis

    pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs pctx1
    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for <t>PcTx1</t> modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology"

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.3001321

    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.
    Figure Legend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.

    Techniques Used: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY, Standard Deviation

    (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.
    Figure Legend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.

    Techniques Used: Expressing, Incubation, Purification, Western Blot, Mutagenesis

    synthetic pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs synthetic pctx1
    (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, highlighted in red. <t>PcTx1</t> (teal) binds to the subunit interfaces. (B) Representative two-electrode voltage clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (C) Same as in (B) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (E) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.
    Synthetic 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
    https://www.bioz.com/result/synthetic pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition"

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    Journal: bioRxiv

    doi: 10.1101/2021.06.21.449215

    (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. (B) Representative two-electrode voltage clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (C) Same as in (B) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (E) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.
    Figure Legend Snippet: (A) Structural overview (PDB ID 4FZO) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labelling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. (B) Representative two-electrode voltage clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9) (C) Same as in (B) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. (D) Concentration-response relationship of WT ASIC1a activation and SSD in absence and presence of 30 nM PcTx1 retrieved form experiments shown in (B) and (C). (E) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). (F) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in B relative to the fluorescence observed upon PcTx1 application. (G) Representative trace of a VCF recording of V80C* equivalent to the ones shown in (E). (H) Same as in (F) but for V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B-C) and 10 μA (E and G) (black vertical), and 10% (red, E and G only). Data in D, F and H are presented as mean + 95CI; n = 3-18 for individual data points in D.

    Techniques Used: Activation Assay, Concentration Assay, Fluorescence

    (A) VCF trace of K105C* showing that the channel readily returns to a functional ‘apo’ state (current, black trace) after application of 300 nM PcTx1 at pH 7.4, while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace). (B) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle) and pH 8.0 (right). (C) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in (B) normalized to the fluorescence change induced by pH 5.5 application. (D) Schematic representation of the different pH dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH in the presence of PcTx1 and leads to conformational rearrangements in both ASIC1a ECD and pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PxTx1 is absent in the extracellular solution. Teal background shading indicates the presence of PcTx1 in the extracellular solution. Transitions between the binding modes that are explicitly shown in this work are indicated in full opacity. (E) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s BigDyn (1 μM) application (purple bar), with subsequent washout and activation. BigDyn is then applied again after the ‘ECD only ’ state is evoked through PcTx1 (300 nM) application, this time only resulting in a slow decrease in the fluorescence signal. (F) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the ‘apo’ (Control) and to the PcTx1-induced ‘ECD only ’ state (Post PcTx1), normalized to the signal induced by pH 5.5; and of the fluorescence signal induced by PcTx1 at pH 7.4 (Control) and BigDyn pre-application (Post BigDyn), respectively. Scale bars are 60 s (black horizontal), 10 μA (black vertical), and 10% (A and E) or 5 % (B) (red, A, B and E only). Data in C and F are presented as mean + 95CI.
    Figure Legend Snippet: (A) VCF trace of K105C* showing that the channel readily returns to a functional ‘apo’ state (current, black trace) after application of 300 nM PcTx1 at pH 7.4, while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace). (B) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle) and pH 8.0 (right). (C) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in (B) normalized to the fluorescence change induced by pH 5.5 application. (D) Schematic representation of the different pH dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH in the presence of PcTx1 and leads to conformational rearrangements in both ASIC1a ECD and pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PxTx1 is absent in the extracellular solution. Teal background shading indicates the presence of PcTx1 in the extracellular solution. Transitions between the binding modes that are explicitly shown in this work are indicated in full opacity. (E) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s BigDyn (1 μM) application (purple bar), with subsequent washout and activation. BigDyn is then applied again after the ‘ECD only ’ state is evoked through PcTx1 (300 nM) application, this time only resulting in a slow decrease in the fluorescence signal. (F) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the ‘apo’ (Control) and to the PcTx1-induced ‘ECD only ’ state (Post PcTx1), normalized to the signal induced by pH 5.5; and of the fluorescence signal induced by PcTx1 at pH 7.4 (Control) and BigDyn pre-application (Post BigDyn), respectively. Scale bars are 60 s (black horizontal), 10 μA (black vertical), and 10% (A and E) or 5 % (B) (red, A, B and E only). Data in C and F are presented as mean + 95CI.

    Techniques Used: Functional Assay, Fluorescence, Binding Assay, Activation Assay

    (A) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a close up of the interaction site at the acidic pocket, including ASIC1a residue F350 (orange). (B) Representative TEVC trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. (C) Activation and SSD curve of F350L mASIC1a without (orange) and with (teal) PcTx1 (n=5–12). (D) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. (E) PcTx1 concentration-response curve at pH 7.4 using the protocol shown in (D) (n=4–14). (F) Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. (G) Left: Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: Comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L (H) Left: Representative trace of a VCF recording of V80C*F350L equivalent to the one shown in G. Right: Same analysis as in G but compared between V80C* and V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B and D only) and 10 μA (black vertical), and 10% (red) (F-H). Data in C, E, G and H are presented as mean + 95CI.
    Figure Legend Snippet: (A) Model of the co-crystal structure of cASIC1a and PcTx1 (teal) binding to the extracellular domain (PDBID 4FZO). Inset shows a close up of the interaction site at the acidic pocket, including ASIC1a residue F350 (orange). (B) Representative TEVC trace showing mASIC1a F350L pH activation in the absence (top) and presence (bottom) of 30 nM PcTx1. (C) Activation and SSD curve of F350L mASIC1a without (orange) and with (teal) PcTx1 (n=5–12). (D) TEVC traces showing the effect of 1 nM PcTx1 on mASIC1a WT (top) and 100 nM PcTx1 on F350L (bottom) applied at pH 7.4. (E) PcTx1 concentration-response curve at pH 7.4 using the protocol shown in (D) (n=4–14). (F) Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.4. (G) Left: Representative VCF trace of the K105C*F350L mutant showing application of 300 nM PcTx1 at pH 7.3. Right: Comparison of the fluorescence change upon PcTx1 application and after a 3 min washout between K105C* and K150C*F350L (H) Left: Representative trace of a VCF recording of V80C*F350L equivalent to the one shown in G. Right: Same analysis as in G but compared between V80C* and V80C*F350L. Scale bars are 60 s (black horizontal), 4 μA (B and D only) and 10 μA (black vertical), and 10% (red) (F-H). Data in C, E, G and H are presented as mean + 95CI.

    Techniques Used: Binding Assay, Activation Assay, Concentration Assay, Mutagenesis, Fluorescence

    (A) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two or all three subunits. (B) Representative TEVC trace of activation. (C) Activation curve from recordings shown in B for the four different concatemeric constructs (n=7–13) (D) Representative TEVC trace of SSD (E) SSD profiles from recordings shown in D (n=4–11). (F) Representative TEVC trace of concentration dependent PcTx1 inhibition at pH 7.4. (G) PcTx1 concentration-response curves from data shown in F (n=4–11). Data points in E-G represent mean and 95CI. All scale bars are 4 μA and 60 s (B and D) or 30 s (F), respectively.
    Figure Legend Snippet: (A) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two or all three subunits. (B) Representative TEVC trace of activation. (C) Activation curve from recordings shown in B for the four different concatemeric constructs (n=7–13) (D) Representative TEVC trace of SSD (E) SSD profiles from recordings shown in D (n=4–11). (F) Representative TEVC trace of concentration dependent PcTx1 inhibition at pH 7.4. (G) PcTx1 concentration-response curves from data shown in F (n=4–11). Data points in E-G represent mean and 95CI. All scale bars are 4 μA and 60 s (B and D) or 30 s (F), respectively.

    Techniques Used: Construct, Mutagenesis, Activation Assay, Concentration Assay, Inhibition

    (A) Representative VCF trace of 300 nM PcTx1 application to a concatemeric construct labelled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. (B) Same as in A but one subunit carries a F350L mutation. (C) Same as in A but with two subunits carrying a F350L mutation. (D) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in A–C. Results from non-concatenated channels are indicated for comparison (shown in light grey). (E) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light grey). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red). In D and E, errors are 95CI, unpaired t-Mann-Whitney test to neighbouring bar on the left, *P<0.05, **P<0.005, ***P<0.0005.
    Figure Legend Snippet: (A) Representative VCF trace of 300 nM PcTx1 application to a concatemeric construct labelled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. (B) Same as in A but one subunit carries a F350L mutation. (C) Same as in A but with two subunits carrying a F350L mutation. (D) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in A–C. Results from non-concatenated channels are indicated for comparison (shown in light grey). (E) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light grey). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red). In D and E, errors are 95CI, unpaired t-Mann-Whitney test to neighbouring bar on the left, *P<0.05, **P<0.005, ***P<0.0005.

    Techniques Used: Construct, Mutagenesis, Fluorescence, MANN-WHITNEY

    Schematic representation of a sideview of ASIC1a ECD and TMD and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view colouring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ conformation with increasing number of F350L containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.
    Figure Legend Snippet: Schematic representation of a sideview of ASIC1a ECD and TMD and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view colouring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ conformation with increasing number of F350L containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

    Techniques Used: Binding Assay, Mutagenesis

    nb linker pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs nb linker pctx1
    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 ) <t>PcTx1-bound</t> 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.
    Nb Linker 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
    https://www.bioz.com/result/nb linker pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nb linker pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) 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

    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: 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:

    ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .
    Figure Legend Snippet: ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .

    Techniques Used: Expressing, Activation Assay, Incubation, Binding Assay, Patch Clamp, Immunofluorescence, Staining, Transfection, Fluorescence, SDS Page, Purification


    Figure Legend Snippet:

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Isolation, Mutagenesis, Magnetic Beads, Affinity Purification, Strep-tag, Software

    pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs pctx1
    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 ) <t>PcTx1-bound</t> 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.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) 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

    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: 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:

    ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .
    Figure Legend Snippet: ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .

    Techniques Used: Expressing, Activation Assay, Incubation, Binding Assay, Patch Clamp, Immunofluorescence, Staining, Transfection, Fluorescence, SDS Page, Purification


    Figure Legend Snippet:

    Techniques Used: Expressing, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Isolation, Mutagenesis, Magnetic Beads, Affinity Purification, Strep-tag, Software

    pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs pctx1
    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for <t>PcTx1</t> modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology"

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    Journal: bioRxiv

    doi: 10.1101/2020.11.24.392498

    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.
    Figure Legend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.

    Techniques Used: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY

    (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower extracellular domain (right insets). Positions that crosslinked to biotin-PcTx1 are coloured red, F352 is marked in orange and positions that did not crosslink are coloured green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 min to form covalent hASIC1a-PcTx1 complexes, which are purified via a C-terminal 1D4-tag on hASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT, untransfected cells (UT) and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356 and 357 in the acidic pocket (coloured red in A, left inset), but not at positions 177, 236, 239, 343 or 351 (coloured green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (right insets in A), WT or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 coloured orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data is representative of three individual experiments, see Figures S12-15 for original blots and crosslinking attempts with Bpa.
    Figure Legend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower extracellular domain (right insets). Positions that crosslinked to biotin-PcTx1 are coloured red, F352 is marked in orange and positions that did not crosslink are coloured green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 min to form covalent hASIC1a-PcTx1 complexes, which are purified via a C-terminal 1D4-tag on hASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT, untransfected cells (UT) and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356 and 357 in the acidic pocket (coloured red in A, left inset), but not at positions 177, 236, 239, 343 or 351 (coloured green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (right insets in A), WT or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 coloured orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data is representative of three individual experiments, see Figures S12-15 for original blots and crosslinking attempts with Bpa.

    Techniques Used: Expressing, Incubation, Purification, Western Blot, Mutagenesis

    modulation by pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    Alomone Labs modulation by pctx1
    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for <t>PcTx1</t> modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.
    Modulation By 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
    https://www.bioz.com/result/modulation by pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    modulation by pctx1 - by Bioz Stars, 2023-02
    86/100 stars

    Images

    1) Product Images from "High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology"

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    Journal: bioRxiv

    doi: 10.1101/2020.11.24.392498

    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.
    Figure Legend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and six ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 min (pink bars) before activation at pH 5.6 (grey bars, 5 sec) and the currents were normalized to the average of two control currents after conditioning at pH 7.6 (black bars; control traces shown in Figure S11). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 min (blue bars) before activation at pH 5.6 (grey bars, 5 sec) and the current was normalized to the average of the four preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± S.D, dashed line indicates 100%, values in Table S5 and S6. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann Whitney test (B) or one-way ANOVA with Tukey’s multiple comparisons test (D). Coloured and black bars in (A) and (C) not to scale.

    Techniques Used: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY

    (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower extracellular domain (right insets). Positions that crosslinked to biotin-PcTx1 are coloured red, F352 is marked in orange and positions that did not crosslink are coloured green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 min to form covalent hASIC1a-PcTx1 complexes, which are purified via a C-terminal 1D4-tag on hASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT, untransfected cells (UT) and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356 and 357 in the acidic pocket (coloured red in A, left inset), but not at positions 177, 236, 239, 343 or 351 (coloured green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (right insets in A), WT or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 coloured orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data is representative of three individual experiments, see Figures S12-15 for original blots and crosslinking attempts with Bpa.
    Figure Legend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower extracellular domain (right insets). Positions that crosslinked to biotin-PcTx1 are coloured red, F352 is marked in orange and positions that did not crosslink are coloured green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 min to form covalent hASIC1a-PcTx1 complexes, which are purified via a C-terminal 1D4-tag on hASIC1a and visualized via Western blotting. (C) Western blot of purified hASIC1a WT, untransfected cells (UT) and variants carrying AzF in the extracellular domain detected using the specified antibodies (AB). Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356 and 357 in the acidic pocket (coloured red in A, left inset), but not at positions 177, 236, 239, 343 or 351 (coloured green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower extracellular domain (right insets in A), WT or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 coloured orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data is representative of three individual experiments, see Figures S12-15 for original blots and crosslinking attempts with Bpa.

    Techniques Used: Expressing, Incubation, Purification, Western Blot, Mutagenesis

    psalmotoxin 1 pctx1  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
    Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    Alomone Labs psalmotoxin 1 pctx1
    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
    https://www.bioz.com/result/psalmotoxin 1 pctx1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psalmotoxin 1 pctx1 - by Bioz Stars, 2023-02
    94/100 stars

    Images

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Alomone Labs pctx1
    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 <t>PcTx1</t> (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 < 0.0001.
    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
    https://www.bioz.com/result/pctx1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pctx1 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    86
    Alomone Labs synthetic pctx1
    ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. <t>PcTx1</t> (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .
    Synthetic 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
    https://www.bioz.com/result/synthetic pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic pctx1 - by Bioz Stars, 2023-02
    86/100 stars
      Buy from Supplier

    86
    Alomone Labs modulation by pctx1
    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for <t>PcTx1</t> modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.
    Modulation By 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
    https://www.bioz.com/result/modulation by pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    modulation by pctx1 - by Bioz Stars, 2023-02
    86/100 stars
      Buy from Supplier

    86
    Alomone Labs nb linker pctx1
    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 ) <t>PcTx1-bound</t> 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.
    Nb Linker 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
    https://www.bioz.com/result/nb linker pctx1/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nb linker pctx1 - by Bioz Stars, 2023-02
    86/100 stars
      Buy from Supplier

    94
    Alomone Labs psalmotoxin 1 pctx1
    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 ) <t>PcTx1-bound</t> 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.
    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
    https://www.bioz.com/result/psalmotoxin 1 pctx1/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    psalmotoxin 1 pctx1 - by Bioz Stars, 2023-02
    94/100 stars
      Buy from Supplier

    Image Search Results


    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 < 0.0001.

    Journal: Redox Biology

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

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend 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 < 0.0001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Cell Culture, Fluorescence

    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 < 0.05, **p < 0.01, ***p < 0.001.

    Journal: Redox Biology

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

    doi: 10.1016/j.redox.2022.102253

    Figure Lengend 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 < 0.05, **p < 0.01, ***p < 0.001.

    Article Snippet: Experiments done on WT neurons, untreated or treated with PcTX1 (Alomone labs, #STP200), were performed using the imaging system consisted of an Axiovert 100 inverted microscope (Zeiss), Polychrome V monochromator (TILL Photonics, Planegg, Germany) and a SensiCam cooled charge-coupled device (PCO, Kelheim, Germany).

    Techniques: Fluorescence

    ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Structural overview (PDB ID 4FZ0) of chicken ASIC1 with positions V80 and K105, which were substituted for cysteine and used for channel labeling, highlighted in red. PcTx1 (teal) binds to the subunit interfaces. ( B ) Representative two-electrode voltage-clamp (TEVC) traces recorded from X. laevis oocytes of WT ASIC1a showing pH sensitivity of activation in the absence (upper panel) and presence (lower panel) of 30 nM PcTx1, added in the resting solution (pH 7.9). Scale bars are 4 µA (vertical) and 60 s (horizontal). ( C ) Same as in ( B ) but for steady-state desensitization (SSD). PcTx1 was applied to solutions of decreasing pH in between application of activating pH 5.6 solution. Scale bars are 4 µA (vertical) and 60 s (horizontal). ( D ) Concentration–response relationship of WT ASIC1a activation and SSD in the absence and presence of 30 nM PcTx1 retrieved form experiments shown in ( B ) and ( C ) (n = 6–18). ( E ) Representative traces of voltage-clamp fluorometry (VCF) recordings of K105C* with the current in black and the fluorescence in red. PcTx1 (300 nM) was washed off for 3 min using pH 7.4 (left) or pH 8.4 (right). Scale bars are 60 s (black horizontal), 10 µA (black vertical), and 10% (red vertical). ( F ) Quantitative analysis of the fluorescence signal at the end of the 3 min washout protocols shown in ( E ) relative to the fluorescence observed upon PcTx1 application. ( G ) Representative trace of a VCF recording of V80C* equivalent to the ones shown in ( E ). Scale bars are 60s (black horizontal), 10 µA (black vertical), and 10% (red vertical).( H ) Same as in ( F ) but for V80C*F350L. Data in ( D ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 1—source data 1. TEVC data from mASIC1a WT of activation and SSD with and without PcTx1, as shown in . Figure 1—source data 2. VCF data from K105C* and V80C* of different PcTx1 washout protocols, as shown in and .

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Labeling, Activation Assay, Concentration Assay, Fluorescence

    ( A ) Representative trace of a VCF recording of K105C* showing application of PcTx1 (300 nM) washed off for 3 min with alternating pHs (20 s pH 7.4, 60 s pH 8.4, 40 s pH 7.4, 60 s pH 8.4) before switching to pH 7.4 again. ( B ) Comparison of the fluorescence of K105C* after a 3 min washout of 300 nM PcTx1 using pH 7.4, 8.4, or a mix of the two as shown in the protocol in ( A ) and . ( C ) Same as in ( A ) but for V80C* and running buffer 7.7 instead of 7.4. ( D ) Same as in ( B ) but for V80C* and base on the protocol shown in ( C ) and . All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( B ) and ( D ) are presented as mean ± 95 CI, ordinary analysis of variance (ANOVA).

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Representative trace of a VCF recording of K105C* showing application of PcTx1 (300 nM) washed off for 3 min with alternating pHs (20 s pH 7.4, 60 s pH 8.4, 40 s pH 7.4, 60 s pH 8.4) before switching to pH 7.4 again. ( B ) Comparison of the fluorescence of K105C* after a 3 min washout of 300 nM PcTx1 using pH 7.4, 8.4, or a mix of the two as shown in the protocol in ( A ) and . ( C ) Same as in ( A ) but for V80C* and running buffer 7.7 instead of 7.4. ( D ) Same as in ( B ) but for V80C* and base on the protocol shown in ( C ) and . All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( B ) and ( D ) are presented as mean ± 95 CI, ordinary analysis of variance (ANOVA).

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Fluorescence

    ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Voltage-clamp fluorometry (VCF) trace of K105C* showing the introduction of the ‘Global’ inhibitory binding mode upon application of 300 nM PcTx1 at pH 7.4. During washout and repeated activation, the channel readily returns to a functional apo state (current, black trace) while the fluorescence change induced by PcTx1 is persistent over multiple ASIC1a activations at pH 5.5 (fluorescence, red trace), characteristic for the ‘ECD only ’ state. ( B ) VCF traces highlighting the fluorescence changes associated with application of PcTx1 at pH 8.0 with subsequent application of pH 5.5 (left), pH 7.4 (middle), and pH 8.0 (right). Respective PcTx1 binding modes are indicated below the traces. ( C ) Quantitative comparison of the fluorescence signal 60 s into the pH 7.4 application at the end of the experiments shown in ( B ) normalized to the fluorescence change induced by pH 5.5 application. ( D ) Schematic representation of the different pH-dependent binding modes of PcTx1: A ‘Loose’ closed state at high pH, a ‘Global’ state that exists at neutral/low pH that leads to conformational rearrangements in the extracellular domain (ECD) and the pore (indicated in orange), and an ‘ECD only ’ state in which the conformational rearrangements are only found in the ECD and that exists at neutral/low pH even when PcTx1 is absent in the extracellular solution. Teal background shading in the ‘Loose’ and ‘Global’ indicates the presence of PcTx1 in the extracellular solution (although not mandatory, see text for details). ( E ) VCF trace of K105C* exposed to pH 5.5, followed by a 60 s big dynorphin (BigDyn) (1 µM) application (purple bar), with subsequent washout and activation. BigDyn is reapplied after the ‘ECD only ’ state has been evoked through PcTx1 (300 nM) application, this time resulting in a smaller decrease in the fluorescence signal. ( F ) Quantitative comparison of the fluorescence change induced by a 60 s BigDyn application to the apo (control) and to the PcTx1-induced ‘ECD only ’ state (post PcTx1), normalized to the signal induced by pH 5.5. ( G ) VCF trace of K105C* where 300 nM PcTx1 is applied to the ‘ECD only ’ state. ( H ) Quantitative analysis of the protocol shown in ( G ) comparing the fluorescence change induced by PcTx1 to the apo state at 7.4 (control) with the PcTx1 application to the ‘ECD only ’ state. All scale bars are 60 s (black horizontal), 10 µA (black vertical), and 5% (red vertical). Data in ( C ), ( F ), and ( H ) are presented as mean ± 95 CI. Figure 2—source data 1. VCF data from mASIC1a K105C* of single and multiple activations during PcTx1 washout, as shown in and . Figure 2—source data 2. VCF data from mASIC1a K105C* of PcTx1 application at pH 8.0 followed by different washout protocols, as seen in . Figure 2—source data 3. VCF data of mASIC1a K105C* of BigDyn and PcTx1 application, as seen in and .

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Binding Assay, Activation Assay, Functional Assay, Fluorescence

    ( A ) Representative VCF trace of K105C* showing application of PcTx1 (300 nM) washout for 3 min before pH 5.5 stimulus. Corresponding binding modes are indicated below. ( B ) Comparison of the final pH 5.5-induced current (I) and fluorescence (ΔF) at pH 7.4 in recordings shown in , where channels undergo three 1 min washouts at pH 7.4, each followed by pH 5.5 stimulus (left) or the protocol shown in ( A ) with a single 3 min washout (right). The final pH 5.5-induced current was normalized to the one at the beginning of the recording, the fluorescence was analyzed right before the final pH 5.5 activation and normalized to the deflection induced by PcTx1. ( C ) Representative VCF trace of K105C* showing pH 7.0 stimuli at various states of the recording. Corresponding binding modes are indicated below. ( D ) Representative trace of a VCF recording of K105C* depicting 30 s pre-conditioning with big dynorphin (BigDyn) (1 μM) and subsequent 30 s PcTx1 (300 nM) application and pH 5.5 activation. ( E ) Quantitative comparison of the fluorescence change induced by 300 nM PcTx1 at pH 7.4 in the apo state (control) and after BigDyn (1 μΜ) application, normalized to the signal induced by pH 5.5. Data in ( B ) and ( E ) are presented as mean ± 95CI.

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Representative VCF trace of K105C* showing application of PcTx1 (300 nM) washout for 3 min before pH 5.5 stimulus. Corresponding binding modes are indicated below. ( B ) Comparison of the final pH 5.5-induced current (I) and fluorescence (ΔF) at pH 7.4 in recordings shown in , where channels undergo three 1 min washouts at pH 7.4, each followed by pH 5.5 stimulus (left) or the protocol shown in ( A ) with a single 3 min washout (right). The final pH 5.5-induced current was normalized to the one at the beginning of the recording, the fluorescence was analyzed right before the final pH 5.5 activation and normalized to the deflection induced by PcTx1. ( C ) Representative VCF trace of K105C* showing pH 7.0 stimuli at various states of the recording. Corresponding binding modes are indicated below. ( D ) Representative trace of a VCF recording of K105C* depicting 30 s pre-conditioning with big dynorphin (BigDyn) (1 μM) and subsequent 30 s PcTx1 (300 nM) application and pH 5.5 activation. ( E ) Quantitative comparison of the fluorescence change induced by 300 nM PcTx1 at pH 7.4 in the apo state (control) and after BigDyn (1 μΜ) application, normalized to the signal induced by pH 5.5. Data in ( B ) and ( E ) are presented as mean ± 95CI.

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Binding Assay, Fluorescence, Activation Assay

    ( A ) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two, or all three subunits. ( B ) Representative two-electrode voltage-clamp (TEVC) trace of activation ( C ) Activation curve from recordings shown in ( B ) for the four different concatemeric constructs (n = 7–13). ( D ) Representative TEVC trace of steady-state desensitization (SSD). ( E ) SSD profiles from recordings shown in ( D ) (n = 4–11). ( F ) Representative TEVC trace of concentration-dependent PcTx1 inhibition at pH 7.4. ( G ) PcTx1 concentration–response curves from data shown in ( F ) (n = 4–11). Scale bars are 4 µA (vertical) and 60 s (horizontal) for (B, D) and 30 s for (F). Data points in ( C, E and G ) represent mean ± 95CI. Figure 4—source data 1. TEVC data from concatemeric mASIC1a of pH-dependent activation, as shown in and . Figure 4—source data 2. TEVC data from concatemeric mASIC1a of SSD, as shown in . Figure 4—source data 3. TEVC data from concatemeric mASIC1a of PcTx1 concentration–response curve, as shown in and .

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Schematic overview of the concatemeric constructs containing the F350L mutation (orange) in none, one, two, or all three subunits. ( B ) Representative two-electrode voltage-clamp (TEVC) trace of activation ( C ) Activation curve from recordings shown in ( B ) for the four different concatemeric constructs (n = 7–13). ( D ) Representative TEVC trace of steady-state desensitization (SSD). ( E ) SSD profiles from recordings shown in ( D ) (n = 4–11). ( F ) Representative TEVC trace of concentration-dependent PcTx1 inhibition at pH 7.4. ( G ) PcTx1 concentration–response curves from data shown in ( F ) (n = 4–11). Scale bars are 4 µA (vertical) and 60 s (horizontal) for (B, D) and 30 s for (F). Data points in ( C, E and G ) represent mean ± 95CI. Figure 4—source data 1. TEVC data from concatemeric mASIC1a of pH-dependent activation, as shown in and . Figure 4—source data 2. TEVC data from concatemeric mASIC1a of SSD, as shown in . Figure 4—source data 3. TEVC data from concatemeric mASIC1a of PcTx1 concentration–response curve, as shown in and .

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Construct, Mutagenesis, Activation Assay, Concentration Assay, Inhibition

    ( A ) Activation and steady-state desensitization (SSD) curves for trimeric and concatemeric WT and F350L channels in comparison. ( B ) Activation curves for all concatemeric variants showing that concatemers with the same number of F350L-bearing subunits cluster around similar pH sensitivities. ( C ) Same as in ( B ), but for concentration-dependent PcTx1 inhibition. Data are presented as mean ± 95 CI.

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Activation and steady-state desensitization (SSD) curves for trimeric and concatemeric WT and F350L channels in comparison. ( B ) Activation curves for all concatemeric variants showing that concatemers with the same number of F350L-bearing subunits cluster around similar pH sensitivities. ( C ) Same as in ( B ), but for concentration-dependent PcTx1 inhibition. Data are presented as mean ± 95 CI.

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Activation Assay, Concentration Assay, Inhibition

    ( A ) Representative voltage-clamp fluorometry (VCF) trace of 300 nM PcTx1 application to a concatemeric construct labeled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. ( B ) Same as in ( A ) but one subunit carries a F350L mutation. ( C ) Same as in ( A ) but with two subunits carry a F350L mutation. ( D ) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in ( A – C ). Results from non-concatenated channels are indicated for comparison (shown in light gray). ( E ) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light gray). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red vertical). In ( D ) and ( E ), error bars represents 95CI, unpaired Mann–Whitney test to neighboring bar on the left, *p<0.05, **p<0.005, ***p<0.0005. Figure 5—source data 1. VCF data from concatemeric mASIC1a of PcTx1 application and washout as shown in .

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: ( A ) Representative voltage-clamp fluorometry (VCF) trace of 300 nM PcTx1 application to a concatemeric construct labeled at K105C* in all three subunits (red star) and subsequent washout for 3 min with pH 7.4 and 40 s pH 8.4. ( B ) Same as in ( A ) but one subunit carries a F350L mutation. ( C ) Same as in ( A ) but with two subunits carry a F350L mutation. ( D ) Comparison of the PcTx1-induced change in the fluorescence signal between the different concatemeric constructs shown in ( A – C ). Results from non-concatenated channels are indicated for comparison (shown in light gray). ( E ) Comparison of the fluorescence intensity after a 3 min washout relative to the intensity upon PcTx1 application. Results from non-concatenated channels are indicated for comparison (shown in light gray). All scale bars represent 10 μA (black vertical), 60 s (black horizontal), 1% (red vertical). In ( D ) and ( E ), error bars represents 95CI, unpaired Mann–Whitney test to neighboring bar on the left, *p<0.05, **p<0.005, ***p<0.0005. Figure 5—source data 1. VCF data from concatemeric mASIC1a of PcTx1 application and washout as shown in .

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Construct, Labeling, Mutagenesis, Fluorescence, MANN-WHITNEY

    Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

    Journal: eLife

    Article Title: Conformational decoupling in acid-sensing ion channels uncovers mechanism and stoichiometry of PcTx1-mediated inhibition

    doi: 10.7554/eLife.73384

    Figure Lengend Snippet: Schematic representation of a side view of acid-sensing ion channel 1a (ASIC1a) extracellular domain (ECD) and transmembrane domain (TMD) and top view of the three subunits and consequences of PcTx1 (teal) binding at neutral/low pH (as in ) and with the F350L mutation (orange) in 0–3 subunits. The side view coloring shows the decreasing stability of the PcTx1-induced ‘ECD only ’ state with increasing number of F350L-containing subunits, and the decreasing inhibitory effect on the pore. In channels with a single F350L subunit, only the PcTx1-induced conformational state of the ECD is affected, while the TMD behaves WT-like.

    Article Snippet: Synthetic PcTx1 was obtained from Alomone Labs (>95% purity).

    Techniques: Binding Assay, Mutagenesis

    (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.

    Journal: PLoS Biology

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    doi: 10.1371/journal.pbio.3001321

    Figure Lengend Snippet: (A) Characteristic current traces and (B) normalized response after SSD in absence or presence of BigDyn for hASIC1a WT and 6 ncAA variants. Cells were incubated at the desensitizing pH specified for each variant with or without 3 μM BigDyn for 2 minutes (pink bars) before activation at pH 5.6 (gray bars, 5 seconds), and the currents were normalized to the average of 2 control currents after conditioning at pH 7.6 (black bars; control traces shown in ). (C) Exemplary current traces and (D) bar graph for PcTx1 modulation of hASIC1a WT and selected variants containing AzF in the acidic pocket at different pH. Cells were incubated with 100 nM PcTx1 at varying pH for 2 minutes (blue bars) before activation at pH 5.6 (gray bars, 5 seconds), and the current was normalized to the average of the 4 preceding and following control currents after conditioning at pH 7.4 (black bars). Bar graphs show mean ± SD, dashed line indicates 100%, and values are shown in and Tables. (*) denotes significant difference between groups, p < 0.05; (**): p < 0.01; (***): p < 0.001; ns: not significant; Mann–Whitney test (B) or 1-way ANOVA with Tukey multiple comparisons test (D). Colored and black bars in (A) and (C) not to scale. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 11, 12, 34, and 35). AzF, 4-Azido-l-phenylalanine; BigDyn, big dynorphin; hASIC1a, human acid-sensing ion channel 1a; ncAA, noncanonical amino acid; PcTx1, psalmotoxin 1; SD, standard deviation; SSD, steady-state desensitization; WT, wild type.

    Article Snippet: To assess modulation by PcTx1 (Alomone Labs, Israel, >95% purity), cells were exposed to 2 control measurements of activation with pH 5.6 after conditioning at pH 7.4 (interval 3.75 minutes), followed by pH 5.6 activation after incubation with 100 nM PcTx1 at varying pH (pH 7.4 to 7.0) for 2 minutes (total interval between stimuli 7 minutes), as well as 2 further controls to assess recovery from modulation.

    Techniques: Incubation, Variant Assay, Activation Assay, MANN-WHITNEY, Standard Deviation

    (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.

    Journal: PLoS Biology

    Article Title: High-throughput characterization of photocrosslinker-bearing ion channel variants to map residues critical for function and pharmacology

    doi: 10.1371/journal.pbio.3001321

    Figure Lengend Snippet: (A) Structure of cASIC1 (white) in complex with PcTx1 (blue, PDB: 4FZ0), insets show individual side chains replaced by AzF in the acidic pocket (left inset) and lower ECD (right insets). Positions that crosslinked to biotin-PcTx1 are colored red, F352 is marked in orange, and positions that did not crosslink are colored green. (B) Schematic workflow for crosslinking to biotin-PcTx1. HEK 293T ASIC1a-KO cells expressing AzF-containing hASIC1a variants are incubated with 100 nM biotin-PcTx1 and exposed to UV light for 15 minutes to form covalent hASIC1a–PcTx1 complexes, which are purified via a carboxyl-terminal 1D4-tag on hASIC1a and visualized via western blotting. (C) Western blot of purified hASIC1a WT, UT cells, and variants carrying AzF in the ECD detected using the specified AB. Biotin-PcTx1 is detected in UV-exposed samples containing AzF at positions 344, 355, 356, and 357 in the acidic pocket (colored red in A, left inset), but not at positions 177, 236, 239, 343, or 351 (colored green in A, left inset). PcTx1 is also absent in control samples not exposed to UV, those carrying AzF in the lower ECD (right insets in A), WT, or UTs. PcTx1 can be detected upon UV-exposing the toxin-insensitive F352L K356AzF double mutant (left inset in A, F352 colored orange). Of note, the anti-biotin AB detects endogenous biotin-dependent carboxylases, which are also found in purified samples from UTs and have been described before [ , ]. Data are representative of 3 individual experiments; see – Figs for original blots and crosslinking attempts with Bpa. The underlying data have been deposited at zenodo.org ( https://doi.org/10.5281/zenodo.4906985 ; files 12 and 13). AB, antibodies; ASIC1a, acid-sensing ion channel 1a; AzF, 4-Azido-l-phenylalanine; Bpa, 4-Benzoyl-l-phenylalanine; cASIC1, chicken acid-sensing ion channel 1; ECD, extracellular domain; hASIC1a, human acid-sensing ion channel 1a; PcTx1, psalmotoxin 1; PDB, Protein Data Bank; UT, untransfected; WT, wild type.

    Article Snippet: To assess modulation by PcTx1 (Alomone Labs, Israel, >95% purity), cells were exposed to 2 control measurements of activation with pH 5.6 after conditioning at pH 7.4 (interval 3.75 minutes), followed by pH 5.6 activation after incubation with 100 nM PcTx1 at varying pH (pH 7.4 to 7.0) for 2 minutes (total interval between stimuli 7 minutes), as well as 2 further controls to assess recovery from modulation.

    Techniques: Expressing, Incubation, Purification, Western Blot, Mutagenesis

    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.

    Journal: eLife

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

    doi: 10.7554/eLife.67115

    Figure Lengend Snippet: 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.

    Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

    Techniques:

    ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .

    Journal: eLife

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

    doi: 10.7554/eLife.67115

    Figure Lengend Snippet: ( 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<0.001. ( D ) Cartoon of the proposed mechanism of how Nb.C1 associated with hASIC1a may interfere with MitTx binding. ( E ) Whole-cell patch clamp of SH-SY5Y cells activated with pH 6.0 generates typical hASIC1a currents. Proton-induced currents are inhibited by PcTx and amiloride. ( F ) Immunofluorescence confocal image of SH-SY5Y cells incubated with Nb.C1-PcTx1-HA fusion and anti-HA antibody (green) shows cells decorated on the periphery. Nuclei were stained with DAPI (blue). Scale bar, 5 µm. ( G ) Cartoon representation showing the Nb.C1-PcTx1 polypeptide binding to two distinct sites on the surface of hASIC1a, accounting for a possible mechanism of toxin potentiation. ( H ) Confocal images of live HEK-293 cells transfected with hASCIC1a-Flag on coverslips incubated with Nb.C1-HA for 30 min and followed for 0, 1, 2, 3, and 4 hr at 18°C in DMEM containing HEPES. Three of the five time points are shown. At each 1 hr interval, all cells were washed except for the one dish of cells removed for fixation. All cells were processed for immunofluorescence with HA and Flag monoclonals to visualize Nb.C1-HA and hASIC1a-Flag, respectively. Nb.C1-HA labels only the cell surface whereas hASIC1a distributes in the plasma membrane and intracellular endoplasmic reticulum and perinuclear membrane. Scale bar, 5 µm. ( I ) Quantification of fluorescence intensity of Nb.C1 (red channel) normalized to time 0 hr ( t 0 ). For each time point 300 cells were analyzed. Columns are the mean ± SEM. ( J ) Coomassie blue SDS-PAGE of purified fusion proteins (Nb.C1-FlexLinker-PcTx and Nb.C1-RigidLinker-PcTx) and Nb.C1 alone. On the right a cartoon representation of the fusion proteins. ( K ) Representative examples of oocytes expressing hASIC1a exposed to 10 nM of PcTx1 or 10 mM of Nb.C1-Rigid-PcTx1 fusion for 60 s prior to serial activations with a change of pH from 7.35 to 6.0. Cells remained in the perfusion chamber throughout the experiment. ( L ) Time course of recovery of acid-induced currents in control (no pretreatment), and pretreatment with PcTx1, Nb.C1-Flex-PcTx, or Nb.C1-Rigid-PcTx1. Preconditioning pH 7.35, activation pH 6.0. Data were fit with a single exponential a ( 1 − e − t / τ ) where τ is 220 s for PcTx, 350 and 880 s for Nb.C1-Flex-PcTx and Nb.C1-Rigid-PcTx; a = 0.90 for PcTx, and 0.16 and 0.14 for the fusions, respectively. Data points represent the mean ± SD of 7–12 cells. Values of currents from each cell are shown in .

    Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

    Techniques: Expressing, Activation Assay, Incubation, Binding Assay, Patch Clamp, Immunofluorescence, Staining, Transfection, Fluorescence, SDS Page, Purification

    Journal: eLife

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

    doi: 10.7554/eLife.67115

    Figure Lengend Snippet:

    Article Snippet: PcTx1 was used at 10 nM and MitTx at 50 nM, both were purchased from Alomone Labs. Oocytes were pre-incubated with purified Nb or Nb-linker-PcTx1 (10 nM) for 20 min at RT before recording.

    Techniques: Expressing, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Isolation, Mutagenesis, Magnetic Beads, Affinity Purification, Strep-tag, Software