anti cav2 1  (Alomone Labs)


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    Structured Review

    Alomone Labs anti cav2 1
    Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP.  (A)  Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm.  (B)  Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n  = 19) and  (C)  in neurites ( n  = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP.  (D)  Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al.,   2015 ).
    Anti Cav2 1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons"

    Article Title: New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00342

    Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP.  (A)  Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm.  (B)  Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n  = 19) and  (C)  in neurites ( n  = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP.  (D)  Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al.,   2015 ).
    Figure Legend Snippet: Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP. (A) Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm. (B) Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n = 19) and (C) in neurites ( n = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP. (D) Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al., 2015 ).

    Techniques Used: Immunoprecipitation

    Ca v 2.1 protein is mis-expressed at the plasma membrane of  Fmr1 -KO cortical neurons.  (A)  Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control.  (B)  Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P
    Figure Legend Snippet: Ca v 2.1 protein is mis-expressed at the plasma membrane of Fmr1 -KO cortical neurons. (A) Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control. (B) Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P

    Techniques Used: Western Blot, Immunoprecipitation, MANN-WHITNEY

    2) Product Images from "Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels"

    Article Title: Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels

    Journal: Frontiers in Synaptic Neuroscience

    doi: 10.3389/fnsyn.2016.00015

    DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.
    Figure Legend Snippet: DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.

    Techniques Used: shRNA, Expressing, Inhibition

    DISC1 enhances Cav2.2 and Cav2.1 currents. (A) Western blot showing expression of ectopic (human) DISC1 in HEK293 cells. (B,C) Cav2.2 Current-voltage (I-V) curves for hDISC1-expressing and control cells. (B) Stimulation protocol and individual current responses shown at three different voltages (−30, 0 and 30 mV) (C) . Average I-V plots for hDISC1 (peak = 54.2 ± 4.5 pA/pF, 13 cells) and control (peak = 39.2 ± 4.9 pA/pF, 12 cells), p = 0.033. (D–F) Cav2.2 activation curves in response to the tail protocol. (D) Illustration of the tail protocol and individual tail currents measured at −50 mV after three different voltage steps (−20, 0 and 40 mV). (E) Average current density based on tail currents for DISC1 (164.3 ± 7.3 pA/pF, 12 cells) and control (110.5 ± 6.3 pA/pF, 12 cells), * p
    Figure Legend Snippet: DISC1 enhances Cav2.2 and Cav2.1 currents. (A) Western blot showing expression of ectopic (human) DISC1 in HEK293 cells. (B,C) Cav2.2 Current-voltage (I-V) curves for hDISC1-expressing and control cells. (B) Stimulation protocol and individual current responses shown at three different voltages (−30, 0 and 30 mV) (C) . Average I-V plots for hDISC1 (peak = 54.2 ± 4.5 pA/pF, 13 cells) and control (peak = 39.2 ± 4.9 pA/pF, 12 cells), p = 0.033. (D–F) Cav2.2 activation curves in response to the tail protocol. (D) Illustration of the tail protocol and individual tail currents measured at −50 mV after three different voltage steps (−20, 0 and 40 mV). (E) Average current density based on tail currents for DISC1 (164.3 ± 7.3 pA/pF, 12 cells) and control (110.5 ± 6.3 pA/pF, 12 cells), * p

    Techniques Used: Western Blot, Expressing, Activation Assay

    3) Product Images from "Alterations of the Sympathoadrenal Axis Related to the Development of Alzheimer’s Disease in the 3xTg Mouse Model"

    Article Title: Alterations of the Sympathoadrenal Axis Related to the Development of Alzheimer’s Disease in the 3xTg Mouse Model

    Journal: Biology

    doi: 10.3390/biology11040511

    Analysis of mRNA levels and the expression of different VDCC subtypes in adrenal medullae of WT and 3xTg mice over 12 m age. ( A ) Normalized mRNA levels of different VDCCs in adrenal medullae from WT and 3xTg mice > 12 m. Both medullas of the same mouse were homogenized, so each dot represents an individual mouse. ( B ) Representative image of WT and 3xTg adrenal medulla stained with anti-Cav1.3 (top) and anti Cav2.1 (bottom) + DAPI (nuclei). ( C ) IntDen quantification of calcium channel expression. Each dot represents an image analyzed, and the bars represent mean ± SEM. A Student’s t -test was used to statistically compare the WT and 3xTg groups. ** p
    Figure Legend Snippet: Analysis of mRNA levels and the expression of different VDCC subtypes in adrenal medullae of WT and 3xTg mice over 12 m age. ( A ) Normalized mRNA levels of different VDCCs in adrenal medullae from WT and 3xTg mice > 12 m. Both medullas of the same mouse were homogenized, so each dot represents an individual mouse. ( B ) Representative image of WT and 3xTg adrenal medulla stained with anti-Cav1.3 (top) and anti Cav2.1 (bottom) + DAPI (nuclei). ( C ) IntDen quantification of calcium channel expression. Each dot represents an image analyzed, and the bars represent mean ± SEM. A Student’s t -test was used to statistically compare the WT and 3xTg groups. ** p

    Techniques Used: Expressing, Mouse Assay, Staining

    4) Product Images from "Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels"

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    Journal: eNeuro

    doi: 10.1523/ENEURO.0081-22.2022

    Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data Fig. 9-1 ).
    Figure Legend Snippet: Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data Fig. 9-1 ).

    Techniques Used:

    Direct binding of Syt-7α to the synprint site of P/Q-type Ca v 2.1 channels. A , B , In brain lysates ( A ) and transfected tsA-201 cells ( B ), co-immunoprecipitation experiments show the binding of Syt-7α and α 1A subunit of Ca v 2.1 channels . C , Binding of Syt-7α (GST-Syt-7α) to the His-tagged synprint site (724–981) of α 1A subunit of P/Q-type Ca v 2.1 channel. GST-Syt-7α were immobilized on glutathione-Sepharose beads and incubated with His-Ca v 2.1 or His-Ca v 1.2 using different Ca 2+ concentrations (10 μ m to 1 m m ). The binding experiments were performed in a Ca 2+ buffering system containing: 5 m m N-hydroxyethyl ethylenediaminetriacetic, 50 m m HEPES (pH 7.2), and 150 m m NaCl. The free Ca 2+ concentrations were estimated using the MAX CHELATOR software. The Ca 2+ concentrations used were 10 μ m , 50 μ m , 100 μ m , 300 μ m , 500 μ m , 700 μ m , and 1 m m . After extensive washing, GST-Syt-7α bound to the beads were eluted with 15 m m reduced glutathione (GSH) in 50 m m Tris-HCl (pH 8) and proceed to electrophoresis and immunoblotting. The bound His-Ca v 2.1 synprint (724–981) proteins were detected with anti-His antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show comparisons clearly. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. A related co-immunoprecipitation experiment conducted with different experimental conditions is presented in Extended Data Figure 1-1 .
    Figure Legend Snippet: Direct binding of Syt-7α to the synprint site of P/Q-type Ca v 2.1 channels. A , B , In brain lysates ( A ) and transfected tsA-201 cells ( B ), co-immunoprecipitation experiments show the binding of Syt-7α and α 1A subunit of Ca v 2.1 channels . C , Binding of Syt-7α (GST-Syt-7α) to the His-tagged synprint site (724–981) of α 1A subunit of P/Q-type Ca v 2.1 channel. GST-Syt-7α were immobilized on glutathione-Sepharose beads and incubated with His-Ca v 2.1 or His-Ca v 1.2 using different Ca 2+ concentrations (10 μ m to 1 m m ). The binding experiments were performed in a Ca 2+ buffering system containing: 5 m m N-hydroxyethyl ethylenediaminetriacetic, 50 m m HEPES (pH 7.2), and 150 m m NaCl. The free Ca 2+ concentrations were estimated using the MAX CHELATOR software. The Ca 2+ concentrations used were 10 μ m , 50 μ m , 100 μ m , 300 μ m , 500 μ m , 700 μ m , and 1 m m . After extensive washing, GST-Syt-7α bound to the beads were eluted with 15 m m reduced glutathione (GSH) in 50 m m Tris-HCl (pH 8) and proceed to electrophoresis and immunoblotting. The bound His-Ca v 2.1 synprint (724–981) proteins were detected with anti-His antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show comparisons clearly. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. A related co-immunoprecipitation experiment conducted with different experimental conditions is presented in Extended Data Figure 1-1 .

    Techniques Used: Binding Assay, Transfection, Immunoprecipitation, Incubation, Software, Electrophoresis, Western Blot, Clarification Assay

    Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.
    Figure Legend Snippet: Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.

    Techniques Used: Transfection, Expressing

    Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.
    Figure Legend Snippet: Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.

    Techniques Used:

    Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.
    Figure Legend Snippet: Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.

    Techniques Used: Transfection

    Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.
    Figure Legend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Techniques Used: Transfection

    Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.
    Figure Legend Snippet: Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

    Techniques Used: Transfection, Expressing

    Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.
    Figure Legend Snippet: Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Techniques Used:

    Syt-7 isoforms α, β, and γ co-immunoprecipitate with Ca v 2.1 channels. A , Left, In transfected tsA-201 cells, co-immunoprecipitation experiments show the binding of Syt-7β and α 1A subunit of Ca v 2.1. Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody. The immunoprecipitates were resolved by SDS/PAGE and immunoblotted with antibody against Syt-7. Right, The reverse experiment where Syt-7β were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. B , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels together with Syt-7γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where Syt-7γ were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. C , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels along with Syt-7α, β, and γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where the three isoforms of Syt-7 were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show specific comparisons. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. Whole-cell voltage clamp experiments show that Ca v 2.1 expressed alone gives similar peak Ba 2+ and Ca 2+ currents as Ca v 2.1 + Syt7-αβγ (Extended Data Fig. 8-1 ).
    Figure Legend Snippet: Syt-7 isoforms α, β, and γ co-immunoprecipitate with Ca v 2.1 channels. A , Left, In transfected tsA-201 cells, co-immunoprecipitation experiments show the binding of Syt-7β and α 1A subunit of Ca v 2.1. Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody. The immunoprecipitates were resolved by SDS/PAGE and immunoblotted with antibody against Syt-7. Right, The reverse experiment where Syt-7β were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. B , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels together with Syt-7γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where Syt-7γ were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. C , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels along with Syt-7α, β, and γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where the three isoforms of Syt-7 were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show specific comparisons. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. Whole-cell voltage clamp experiments show that Ca v 2.1 expressed alone gives similar peak Ba 2+ and Ca 2+ currents as Ca v 2.1 + Syt7-αβγ (Extended Data Fig. 8-1 ).

    Techniques Used: Transfection, Immunoprecipitation, Binding Assay, SDS Page, Western Blot, Clarification Assay

    Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.
    Figure Legend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

    Techniques Used: Activation Assay, Expressing

    Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data Fig. 10-1 ).
    Figure Legend Snippet: Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data Fig. 10-1 ).

    Techniques Used:

    5) Product Images from "The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels"

    Article Title: The sorting receptor Rer1 controls Purkinje cell function via voltage gated sodium channels

    Journal: Scientific Reports

    doi: 10.1038/srep41248

    Absence of Rer1 reduces Na v  protein levels. ( a ) Brain lysates of Rer1 Δbrain  (ko) and control littermates (wt) of different ages as indicated were separated by SDS-PAGE, blotted and probed with indicated antibodies. In the Ca v 2.1 blot the irrelevant lane #7 was removed and the two last lanes flipped vertically, to match loading of the other gels. See full-length blot in the  supplemental Fig. 5 . P38, synaptophysin. ( b ) De-glycosylation of Na v 1.1 and 1.6 indicates maturation status of the different bands. Brain lysates of P15 wt mice were subjected to de-glycosylation with endoglycosidase H (H) or PNGaseF (F) and processed for Western Blotting with Na v 1.1 and 1.6 antibodies. Im, immature (endoH sensitive); m, mature (endoH resistant); degl, de-glycosylated Na v 1.1 or 1.6 after PNGase F digest. In a, b cropped blots are shown, full-length blots are shown in  supplemental Figs 5,6 .
    Figure Legend Snippet: Absence of Rer1 reduces Na v protein levels. ( a ) Brain lysates of Rer1 Δbrain (ko) and control littermates (wt) of different ages as indicated were separated by SDS-PAGE, blotted and probed with indicated antibodies. In the Ca v 2.1 blot the irrelevant lane #7 was removed and the two last lanes flipped vertically, to match loading of the other gels. See full-length blot in the supplemental Fig. 5 . P38, synaptophysin. ( b ) De-glycosylation of Na v 1.1 and 1.6 indicates maturation status of the different bands. Brain lysates of P15 wt mice were subjected to de-glycosylation with endoglycosidase H (H) or PNGaseF (F) and processed for Western Blotting with Na v 1.1 and 1.6 antibodies. Im, immature (endoH sensitive); m, mature (endoH resistant); degl, de-glycosylated Na v 1.1 or 1.6 after PNGase F digest. In a, b cropped blots are shown, full-length blots are shown in supplemental Figs 5,6 .

    Techniques Used: SDS Page, Mouse Assay, Western Blot

    6) Product Images from "New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons"

    Article Title: New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00342

    Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP.  (A)  Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm.  (B)  Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n  = 19) and  (C)  in neurites ( n  = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP.  (D)  Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al.,   2015 ).
    Figure Legend Snippet: Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP. (A) Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm. (B) Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n = 19) and (C) in neurites ( n = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP. (D) Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al., 2015 ).

    Techniques Used: Immunoprecipitation

    Ca v 2.1 protein is mis-expressed at the plasma membrane of  Fmr1 -KO cortical neurons.  (A)  Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control.  (B)  Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P
    Figure Legend Snippet: Ca v 2.1 protein is mis-expressed at the plasma membrane of Fmr1 -KO cortical neurons. (A) Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control. (B) Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P

    Techniques Used: Western Blot, Immunoprecipitation, MANN-WHITNEY

    7) Product Images from "Fluoxetine Suppresses Glutamate- and GABA-Mediated Neurotransmission by Altering SNARE Complex"

    Article Title: Fluoxetine Suppresses Glutamate- and GABA-Mediated Neurotransmission by Altering SNARE Complex

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20174247

    Inhibition of P/Q type Ca 2+ channels mediates FLX-induced suppression of presynaptic activity. ( A ) Statistical analysis of KCl-evoked syt1-L ab uptake from cortical neurons treated with CTRL solution or FLX (100 µM: 90 min) in the presence or absence of CaV2.1 channels antagonist (AgaTx; 0.4 µM) ( n = 25 CTRL cells; n = 20 FLX; n = 28 AgaTx; n = 25 AgaTx/FLX). ( B ) Representative images of CTRL and FLX-treated cells stained with antibody against CaV2.1. ( C ) Quantification of ( B ) ( n = 31 CTRL cells; n = 32 FLX-treated cells). ( D ) Schematic description and quantification ( E ) of KCl-evoked syt1-L ab uptake along 20 µm of proximal dendrite in cells treated for 90 min with CTRL solution or 100 µM FLX kept during the treatment in normal medium or Ca 2+ -free medium ( n = 29 CTRL; n = 23 FLX; n = 29 no Ca 2+ ; n = 29 no Ca 2+ /FLX). ( F ) Schematic description and statistical analysis (G) of KCl-driven syt1-L ab uptake from control ( n = 29) and FLX-treated cortical neurons ( n = 24) upon chelation of intracellular calcium by BAPTA-AM (10 µM) ( n = 22 BAPTA-AM; n = 15 BAPTA-AM/FLX). In all graphs, values are expressed as means ± SEM. Statistic was done using two-way ANOVA followed by Bonferroni multiple comparison test ( A , E , G ) or Student’s t -test ( C ); * p
    Figure Legend Snippet: Inhibition of P/Q type Ca 2+ channels mediates FLX-induced suppression of presynaptic activity. ( A ) Statistical analysis of KCl-evoked syt1-L ab uptake from cortical neurons treated with CTRL solution or FLX (100 µM: 90 min) in the presence or absence of CaV2.1 channels antagonist (AgaTx; 0.4 µM) ( n = 25 CTRL cells; n = 20 FLX; n = 28 AgaTx; n = 25 AgaTx/FLX). ( B ) Representative images of CTRL and FLX-treated cells stained with antibody against CaV2.1. ( C ) Quantification of ( B ) ( n = 31 CTRL cells; n = 32 FLX-treated cells). ( D ) Schematic description and quantification ( E ) of KCl-evoked syt1-L ab uptake along 20 µm of proximal dendrite in cells treated for 90 min with CTRL solution or 100 µM FLX kept during the treatment in normal medium or Ca 2+ -free medium ( n = 29 CTRL; n = 23 FLX; n = 29 no Ca 2+ ; n = 29 no Ca 2+ /FLX). ( F ) Schematic description and statistical analysis (G) of KCl-driven syt1-L ab uptake from control ( n = 29) and FLX-treated cortical neurons ( n = 24) upon chelation of intracellular calcium by BAPTA-AM (10 µM) ( n = 22 BAPTA-AM; n = 15 BAPTA-AM/FLX). In all graphs, values are expressed as means ± SEM. Statistic was done using two-way ANOVA followed by Bonferroni multiple comparison test ( A , E , G ) or Student’s t -test ( C ); * p

    Techniques Used: Inhibition, Activity Assay, Staining

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    Alomone Labs anti cav2 1
    Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP.  (A)  Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm.  (B)  Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n  = 19) and  (C)  in neurites ( n  = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP.  (D)  Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al.,   2015 ).
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    Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP.  (A)  Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm.  (B)  Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n  = 19) and  (C)  in neurites ( n  = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP.  (D)  Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al.,   2015 ).

    Journal: Frontiers in Molecular Neuroscience

    Article Title: New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons

    doi: 10.3389/fnmol.2018.00342

    Figure Lengend Snippet: Endogenous Ca v 2.1 interacts and partially co-localizes with FMRP. (A) Single plane confocal analysis of FMRP (revealed with the 1C3 antibody) and Ca v 2.1 (revealed with the antibody anti-Ca v 2.1) localization in DIV 13 primary neuronal cultures. The scale bar of each panel is 50 μm. (B) Quantification of the colocalization of FMRP and Ca v 2.1 in the soma ( n = 19) and (C) in neurites ( n = 14) was performed with the JACoP plugin for ImageJ. CC, correlation coefficient; M1, fraction of FMRP overlapping with Ca v 2.1; M2, fraction of Ca v 2.1 overlapping with FMRP. (D) Endogeneous FMRP co-immunoprecipitation with Ca v 2.1 in mouse cerebellar extracts. FMRP was revealed with the 1R antibody (Bonaccorso et al., 2015 ).

    Article Snippet: Primary antibodies anti ß-Actin (Sigma, #A5441; 1/1,000), anti-Cav2.1 (Alomone Labs, #ACC-001; 1/1,000) and anti ß3-tubulin (Synaptic Systems, #302302; 1/1,000) were used.

    Techniques: Immunoprecipitation

    Ca v 2.1 protein is mis-expressed at the plasma membrane of  Fmr1 -KO cortical neurons.  (A)  Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control.  (B)  Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P

    Journal: Frontiers in Molecular Neuroscience

    Article Title: New Insights Into the Role of Cav2 Protein Family in Calcium Flux Deregulation in Fmr1-KO Neurons

    doi: 10.3389/fnmol.2018.00342

    Figure Lengend Snippet: Ca v 2.1 protein is mis-expressed at the plasma membrane of Fmr1 -KO cortical neurons. (A) Western blot analysis of biotinylated Ca v 2.1 in DIV 15–19 cortical neurons. β-tubulin is used as the loading control, whereas actin is used as the immunoprecipitation control. (B) Quantification of total and cell-surface Ca v 2.1 protein levels. Results are presented as the mean ± SEM, Mann-Whitney test: * P

    Article Snippet: Primary antibodies anti ß-Actin (Sigma, #A5441; 1/1,000), anti-Cav2.1 (Alomone Labs, #ACC-001; 1/1,000) and anti ß3-tubulin (Synaptic Systems, #302302; 1/1,000) were used.

    Techniques: Western Blot, Immunoprecipitation, MANN-WHITNEY

    DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fnsyn.2016.00015

    Figure Lengend Snippet: DISC1 regulates Cav2.2-dependent SV exocytosis. (A) Average vGpH traces in scr (101 boutons, 6 fields, 2 exps) and DISC1-E (87 boutons, 5 fields, 2 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.2 blocker ω-Conotoxin GVIA (125 nM). (B) Boxplot of SV exocytic rates before and after ω-Conotoxin GVIA application. Exocytic rates were measured by linear fitting of the first six time points of the vGpH response. (C) Average vGpH traces in scr (1294 boutons, 9 fields, 3 exps) and DISC1-E (1282 boutons, 9 fields, 3 exps) shRNA-expressing neurons during consecutive trains of APs (300 AP, 10 Hz) in the absence or presence of the Cav2.1 blocker ω-Agatoxin TK (125 nM). (D) Boxplot of SV exocytic rates before and after ω-Agatoxin TK application. (E) Table showing the percentage of inhibition of exocytosis rate by DISC1 knockdown before and after Cav2.2- or Cav2.1 blockade.

    Article Snippet: The rabbit polyclonal Abs against Cav2.1 (#ACC-001) and Cav2.2 (#ACC-002) were from Alomone Labs.

    Techniques: shRNA, Expressing, Inhibition

    DISC1 enhances Cav2.2 and Cav2.1 currents. (A) Western blot showing expression of ectopic (human) DISC1 in HEK293 cells. (B,C) Cav2.2 Current-voltage (I-V) curves for hDISC1-expressing and control cells. (B) Stimulation protocol and individual current responses shown at three different voltages (−30, 0 and 30 mV) (C) . Average I-V plots for hDISC1 (peak = 54.2 ± 4.5 pA/pF, 13 cells) and control (peak = 39.2 ± 4.9 pA/pF, 12 cells), p = 0.033. (D–F) Cav2.2 activation curves in response to the tail protocol. (D) Illustration of the tail protocol and individual tail currents measured at −50 mV after three different voltage steps (−20, 0 and 40 mV). (E) Average current density based on tail currents for DISC1 (164.3 ± 7.3 pA/pF, 12 cells) and control (110.5 ± 6.3 pA/pF, 12 cells), * p

    Journal: Frontiers in Synaptic Neuroscience

    Article Title: Stimulation of Synaptic Vesicle Exocytosis by the Mental Disease Gene DISC1 is Mediated by N-Type Voltage-Gated Calcium Channels

    doi: 10.3389/fnsyn.2016.00015

    Figure Lengend Snippet: DISC1 enhances Cav2.2 and Cav2.1 currents. (A) Western blot showing expression of ectopic (human) DISC1 in HEK293 cells. (B,C) Cav2.2 Current-voltage (I-V) curves for hDISC1-expressing and control cells. (B) Stimulation protocol and individual current responses shown at three different voltages (−30, 0 and 30 mV) (C) . Average I-V plots for hDISC1 (peak = 54.2 ± 4.5 pA/pF, 13 cells) and control (peak = 39.2 ± 4.9 pA/pF, 12 cells), p = 0.033. (D–F) Cav2.2 activation curves in response to the tail protocol. (D) Illustration of the tail protocol and individual tail currents measured at −50 mV after three different voltage steps (−20, 0 and 40 mV). (E) Average current density based on tail currents for DISC1 (164.3 ± 7.3 pA/pF, 12 cells) and control (110.5 ± 6.3 pA/pF, 12 cells), * p

    Article Snippet: The rabbit polyclonal Abs against Cav2.1 (#ACC-001) and Cav2.2 (#ACC-002) were from Alomone Labs.

    Techniques: Western Blot, Expressing, Activation Assay

    Analysis of mRNA levels and the expression of different VDCC subtypes in adrenal medullae of WT and 3xTg mice over 12 m age. ( A ) Normalized mRNA levels of different VDCCs in adrenal medullae from WT and 3xTg mice > 12 m. Both medullas of the same mouse were homogenized, so each dot represents an individual mouse. ( B ) Representative image of WT and 3xTg adrenal medulla stained with anti-Cav1.3 (top) and anti Cav2.1 (bottom) + DAPI (nuclei). ( C ) IntDen quantification of calcium channel expression. Each dot represents an image analyzed, and the bars represent mean ± SEM. A Student’s t -test was used to statistically compare the WT and 3xTg groups. ** p

    Journal: Biology

    Article Title: Alterations of the Sympathoadrenal Axis Related to the Development of Alzheimer’s Disease in the 3xTg Mouse Model

    doi: 10.3390/biology11040511

    Figure Lengend Snippet: Analysis of mRNA levels and the expression of different VDCC subtypes in adrenal medullae of WT and 3xTg mice over 12 m age. ( A ) Normalized mRNA levels of different VDCCs in adrenal medullae from WT and 3xTg mice > 12 m. Both medullas of the same mouse were homogenized, so each dot represents an individual mouse. ( B ) Representative image of WT and 3xTg adrenal medulla stained with anti-Cav1.3 (top) and anti Cav2.1 (bottom) + DAPI (nuclei). ( C ) IntDen quantification of calcium channel expression. Each dot represents an image analyzed, and the bars represent mean ± SEM. A Student’s t -test was used to statistically compare the WT and 3xTg groups. ** p

    Article Snippet: After the generous washing of the tissues with PBS and the blocking of the tissues with goat serum, the slices were incubated overnight with primary antibodies—antiCav1.3 (1:200; ACC-003; Alomone Labs; Jerusalem, Israel) and antiCav2.1 (1:200; ACC-001; Alomone labs)—and then with secondary antibodies (1:500).

    Techniques: Expressing, Mouse Assay, Staining

    Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data Fig. 9-1 ).

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data Fig. 9-1 ).

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques:

    Direct binding of Syt-7α to the synprint site of P/Q-type Ca v 2.1 channels. A , B , In brain lysates ( A ) and transfected tsA-201 cells ( B ), co-immunoprecipitation experiments show the binding of Syt-7α and α 1A subunit of Ca v 2.1 channels . C , Binding of Syt-7α (GST-Syt-7α) to the His-tagged synprint site (724–981) of α 1A subunit of P/Q-type Ca v 2.1 channel. GST-Syt-7α were immobilized on glutathione-Sepharose beads and incubated with His-Ca v 2.1 or His-Ca v 1.2 using different Ca 2+ concentrations (10 μ m to 1 m m ). The binding experiments were performed in a Ca 2+ buffering system containing: 5 m m N-hydroxyethyl ethylenediaminetriacetic, 50 m m HEPES (pH 7.2), and 150 m m NaCl. The free Ca 2+ concentrations were estimated using the MAX CHELATOR software. The Ca 2+ concentrations used were 10 μ m , 50 μ m , 100 μ m , 300 μ m , 500 μ m , 700 μ m , and 1 m m . After extensive washing, GST-Syt-7α bound to the beads were eluted with 15 m m reduced glutathione (GSH) in 50 m m Tris-HCl (pH 8) and proceed to electrophoresis and immunoblotting. The bound His-Ca v 2.1 synprint (724–981) proteins were detected with anti-His antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show comparisons clearly. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. A related co-immunoprecipitation experiment conducted with different experimental conditions is presented in Extended Data Figure 1-1 .

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Direct binding of Syt-7α to the synprint site of P/Q-type Ca v 2.1 channels. A , B , In brain lysates ( A ) and transfected tsA-201 cells ( B ), co-immunoprecipitation experiments show the binding of Syt-7α and α 1A subunit of Ca v 2.1 channels . C , Binding of Syt-7α (GST-Syt-7α) to the His-tagged synprint site (724–981) of α 1A subunit of P/Q-type Ca v 2.1 channel. GST-Syt-7α were immobilized on glutathione-Sepharose beads and incubated with His-Ca v 2.1 or His-Ca v 1.2 using different Ca 2+ concentrations (10 μ m to 1 m m ). The binding experiments were performed in a Ca 2+ buffering system containing: 5 m m N-hydroxyethyl ethylenediaminetriacetic, 50 m m HEPES (pH 7.2), and 150 m m NaCl. The free Ca 2+ concentrations were estimated using the MAX CHELATOR software. The Ca 2+ concentrations used were 10 μ m , 50 μ m , 100 μ m , 300 μ m , 500 μ m , 700 μ m , and 1 m m . After extensive washing, GST-Syt-7α bound to the beads were eluted with 15 m m reduced glutathione (GSH) in 50 m m Tris-HCl (pH 8) and proceed to electrophoresis and immunoblotting. The bound His-Ca v 2.1 synprint (724–981) proteins were detected with anti-His antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show comparisons clearly. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. A related co-immunoprecipitation experiment conducted with different experimental conditions is presented in Extended Data Figure 1-1 .

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Binding Assay, Transfection, Immunoprecipitation, Incubation, Software, Electrophoresis, Western Blot, Clarification Assay

    Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Transfection, Expressing

    Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques:

    Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Transfection

    Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Transfection

    Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Transfection, Expressing

    Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques:

    Syt-7 isoforms α, β, and γ co-immunoprecipitate with Ca v 2.1 channels. A , Left, In transfected tsA-201 cells, co-immunoprecipitation experiments show the binding of Syt-7β and α 1A subunit of Ca v 2.1. Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody. The immunoprecipitates were resolved by SDS/PAGE and immunoblotted with antibody against Syt-7. Right, The reverse experiment where Syt-7β were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. B , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels together with Syt-7γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where Syt-7γ were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. C , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels along with Syt-7α, β, and γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where the three isoforms of Syt-7 were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show specific comparisons. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. Whole-cell voltage clamp experiments show that Ca v 2.1 expressed alone gives similar peak Ba 2+ and Ca 2+ currents as Ca v 2.1 + Syt7-αβγ (Extended Data Fig. 8-1 ).

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7 isoforms α, β, and γ co-immunoprecipitate with Ca v 2.1 channels. A , Left, In transfected tsA-201 cells, co-immunoprecipitation experiments show the binding of Syt-7β and α 1A subunit of Ca v 2.1. Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody. The immunoprecipitates were resolved by SDS/PAGE and immunoblotted with antibody against Syt-7. Right, The reverse experiment where Syt-7β were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. B , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels together with Syt-7γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where Syt-7γ were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. C , Left, In tsA-201 cells co-transfected with the α 1A subunit of Ca v 2.1 channels along with Syt-7α, β, and γ, Ca v 2.1 channels were immunoprecipitated with an anti-Ca v 2.1 antibody and blotted with anti-Syt antibody. Right, Reverse experiment where the three isoforms of Syt-7 were immunoprecipitated with an anti-Syt antibody and blotted with anti-Ca v 2.1 antibody. Because different Ca 2+ concentrations were used in co-immunoprecipitation experiments, segments from different immunoblots were spliced together to show specific comparisons. Those protein bands are delineated for clarification. The immunoblots presented here are representative of at least three experiments for each co-immunoprecipitation or immunoblot. Whole-cell voltage clamp experiments show that Ca v 2.1 expressed alone gives similar peak Ba 2+ and Ca 2+ currents as Ca v 2.1 + Syt7-αβγ (Extended Data Fig. 8-1 ).

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Transfection, Immunoprecipitation, Binding Assay, SDS Page, Western Blot, Clarification Assay

    Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: Activation Assay, Expressing

    Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data Fig. 10-1 ).

    Journal: eNeuro

    Article Title: Synaptotagmin-7 Enhances Facilitation of Cav2.1 Calcium Channels

    doi: 10.1523/ENEURO.0081-22.2022

    Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data Fig. 10-1 ).

    Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Cav2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

    Techniques: