Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: ( Top ) Topology of the voltage-gated Cav2.1 subunit with the position of the four pathogenic variants studied: A405T, R1359W, R1667W, and S1799L. The Cavα is formed of four repeats (I to IV) each containing 6 transmembrane helices (S1–S6). The S1–S4 helices constitute the voltage-sensing domain (VSD) able to move in response to membrane potential changes. The four re-entrant P loops between segments S5 and S6 carry the selectivity filter and delineate the extracellular end of the channel pore. The S5 and S6 helices of the four repeats delineate the cytoplasmic end of the channel pore. ( Bottom ) Amino-acid sequences of the I-II loop, and of the IIIS4, IVS4, and IVS6 helices. Cav2.1 variants referenced in the NCBI ClinVar database are indicated with their accession number. References in italics correspond to variants ‘of uncertain significance’ when subjected to in silico analysis. The ‘R’ above the sequences of the IIIS4 and IVS4 helices indicates the position of a basic residue. The Cav2.1 sequence is numbered according to the NCBI Genbank ® sequence AAB64179.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: In Silico, Sequencing

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: ( A ) Representative current traces were recorded from X. laevis oocytes that expressed the indicated Cav2.1+e47 variants with Cavβ4a and Cavα2δ1. Currents were elicited from a holding potential of −100 mV by a two-pulse protocol illustrated on top, and consisting of a 2.5 s-long depolarization from −80 mV to +40 mV, followed by a 400 ms-long depolarization to 0 mV (WT and R1359W) or +10 mV (A405T, R1667W, and S1799L). Scale bars, 200nA. ( B ) Representative images of HEK cells immunostained with anti-Cav2.1 (red) and anti-ZO-1 (green). Anti-ZO-1 was used to visualize the cell membrane. Scale bars, 10 μm. ( C ) Quantification of fluorescence intensity realized on individualized cells ( n = 10 for each variant). Asterisks and number signs denote significant differences vs. WT (*** p < 0.001) and vs. Cav2.1+e47 splice variants (### p < 0.001), respectively (non-paired Student’s t -test). n.s. = non-significant.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Fluorescence, Variant Assay

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Voltage dependence of Cav2.1 activation. ( Top ) Average current-voltage relationships for X. laevis oocytes expressing Cav2.1+47 ( left ) or Cav2.1−e47 variants ( right ). ( Bottom ) Box plots of half-maximal activation potential (Va, left) and slope factor of the Boltzmann curve of channel activation (ka, right) were obtained for all Cav2.1 variants studied. The mean values are given in . Asterisks and number signs denote significant differences vs. WT (** p < 0.01, *** p < 0.001) and vs. Cav2.1+e47 splice variant (# p < 0.05), respectively (non-paired Student’s t -test). n.s. = non-significant.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Activation Assay, Expressing, Variant Assay

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Voltage dependence of Cav2.1 activation and inactivation.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Activation Assay

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Voltage dependence of Cav2.1 inactivation. ( Top ) Voltage-dependent isochronal inactivation curves for X. laevis oocytes expressing Cav2.1+47 ( left ) or Cav2.1−e47 ( right ) variants. ( Bottom ) Box plots of half-maximal inactivation potential (Vi, left) and slope factor of the Boltzmann curve of channel inactivation (ki, right) were obtained for all Cav2.1 variants studied. The mean values are given in . Asterisks and number signs denote significant differences vs. WT (** p < 0.01, *** p < 0.001) and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t -test). n.s. = non-significant.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Expressing

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Cav2.1 inactivation kinetics. Box plot of the ratio of remaining current at the end of a 400 ms-long depolarization (R400) to 0 mV (WT, A405T, R1359W, and R1667W Cav2.1 variants) or + 10 mV (S1799L Cav2.1 variants) with respect to the peak current amplitude. Asterisks and number signs denote significant differences vs. WT (** p < 0.01, *** p < 0.001), and vs. Cav2.1+e47 (# p < 0.05, ### p < 0.001), respectively (non-paired Student’s t -test). n.s. = non-significant.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques:

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Recovery from inactivation of Cav2.1 variants. ( A ) Representative current traces were obtained in X. laevis oocytes expressing Cav2.1+47 with Cavβ4a and Cavα2δ1. The experimental protocol, illustrated above traces, consists of a 2.5 s-long depolarization to +10 mV, followed by inter-pulse intervals between 100 ms and 8 s, and a second 100 ms-long depolarization to +10 mV. Scale bars: 200 nA and 2 s. ( B ) Percentage of current recovery plotted against the inter-pulse duration for Cav2.1+e47 ( top ) and Cav2.1−e47 variants ( bottom ). ( C ) Bar graph showing percent recovery at 8 s for all Cav2.1 variants studied. Asterisks and number signs denote significant differences vs. WT (** p < 0.01, *** p < 0.01) and vs. Cav2.1+e47 (## p < 0.01), respectively (non-paired Student’s t -test). n.s. = non-significant.

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Expressing

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Homology model of human Cav2.1 viewed from the side ( A ) or from the top ( B ). The IIIS4, IVS4, and IVS6 transmembrane helices carrying the studied mutation are represented in magenta, dark green, and brown, respectively. The four residues R1359 (IIIS4), R1667 (IVS4), S1799L (IVS6), and A405 (I-II loop, indicated with black arrow) are represented in balls. ( C , D ) Modeling of the voltage sensing domain (VSD) in repeat III of WT Cav2.1 ( C ), and Cav2.1 R1359W ( D ), showing putative non-bonded interactions in dash lines: hydrogen bond pairs in green (K1355 in IIIS4), ionic bond pairs in yellow (E1291 in IIIS2, and to a lesser extent, D1317 in IIIS3), hydrophobic interaction in purple (L1251 in IIIS1). Steric clashes (K1358 in IIIS4) are represented by a red dashed line. ( E , F ) Modeling of the IV-VSD of WT Cav2.1 ( E ), and Cav2.1 R1667W ( F ). ( G , H ) Modeling of the pore domain around the IVS6 helix of WT Cav2.1 ( G ), and Cav2.1 S1799L ( H ).

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques: Mutagenesis

Journal: Membranes

Article Title: Functional Characterization of Four Known Cav2.1 Variants Associated with Neurodevelopmental Disorders

doi: 10.3390/membranes13010096

Figure Lengend Snippet: Computer modeling of human Purkinje cell. ( A ) Representative firing patterns obtained from simulated Purkinje cell with WT Cav2.1 properties (Va, ka, run 1), with a hyperpolarizing shift of Va (run 2), a depolarization shift of Va (run 3), an increase of ka (run 4), or a combination of both (run 5 and 6). ( B ) Bar graph showing the mean action potential frequencies (freq) obtained for the different simulations (in Hertz: 57.3 ± 0.8, 47.3 ± 1.0, 86.3 ± 0.7, 55.7 ± 0.9, 43.8 ± 0.8 and 85.5 ± 0.7 for run 1 to run 6, respectively). ( C ) Bar graph showing the mean action potential areas obtained for the different simulations (in mV.ms: 64.0 ± 0.1, 64.7 ± 0.2, 62.2 ± 0.1, 64.0 ± 0.1, 64.8 ± 0.2, 57.6 ± 0.1 for run 1 to 6, respectively). The number of action potentials analyzed was between 389 and 807. Asterisks denote significant difference vs. run 1 (** p < 0.01, *** p < 0.01) (non-paired Student’s t -test).

Article Snippet: They were then incubated with anti-Cav2.1 (1:100, ACC-001, Alomone, Jerusalem, Israel), and anti-ZO-1 (1:100, Thermo Fisher Scientific, Waltham, MA, USA) antibodies at 4 °C overnight, washed twice with PBS 1X and then incubated with appropriate secondary antibodies for 1 h at room temperature.

Techniques:

Journal: PLoS ONE

Article Title: Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

doi: 10.1371/journal.pone.0080294

Figure Lengend Snippet: 5 µm-thick formalin-fixed, paraffin-embedded serial sections of 11 week post-conception human fetal lungs were dewaxed and used for immunohistochemistry. A: Expression of P/Q type, Ca v 2.1, and of T-type, Ca v 3.2, calcium channels could be detected at the basolateral side of epithelial cells and in smooth muscle cells, visualised using DAB (brown staining). Scale bar = 5000 µm. B,C: Higher magnification images (40x and 100x) show little-to-no expression of the N-type calcium channel, Ca v 2.2 or the T-type, Ca v 3.3 in the lung parenchyma. Negative controls were carried out through the substitution of the primary antibody with an isotype control. Sections were counterstained with Harris’ hematoxylin (blue staining). Scale bar = 1000 µm.

Article Snippet: Primary antibody suppliers and dilutions used were : Cav1.2 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/50) Cav1.3 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/50) Cav2.1 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/100) Cav2.2 - rabbit polyclonal (Millipore; mouse: 1/100, human: 1/100) Cav2.3 - rabbit polyclonal (Abcam; mouse: 1/100, human: 1/100) Cav3.2 - goat polyclonal (N-18) (Santa Cruz; mouse: 1/100, human:1/100) Cav3.3 - goat polyclonal (N-20) (Santa Cruz; mouse: 1/100, human:1/100) Secondary antibody suppliers and dilutions were: goat anti-rabbit horse radish peroxidase (HRP) (Cav 1.2, 1.3, 2.1, 2.2, and 2.3; DAKO, Ely, U.K.; 1:200 for both mouse and human) or donkey anti-goat HRP (Cav 3.2 and 3.3; Abcam; 1:200 for both mouse and human).

Techniques: Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Expressing, Staining

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Activation Assay, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 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 ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: a Representative confocal image of a nerve terminal arborization. Singly, dually, and innervated by three or more axons NMJs from YFP muscles and also images of the morphologic maturation (S1, the most inmature, and S4, almost fully differentiated, stages) of the postsynaptic clusters from P9 mice. The bar indicates 10 μm. b Confocal immunofluorescence location of α 1D L-, N-, and P/Q-type voltage-dependent calcium channels (VDCCs) at the NMJ. Triple labeling of VDCCs (green fluorescence) with syntaxin (blue fluorescence) and nAChR-α-bungarotoxin (red fluorescence) in merge images. Figure shows the presence of α 1D L-, N-, and P/Q-type-VDCC (in green) in the nerve terminal of P9 Levator auris longus (LAL) muscle endplates. The bar indicates 10 μm

Article Snippet: Muscles were incubated overnight at 4 °C with anti-Ca V 1.3 (CACNA1D) antibody voltage-dependent L-type calcium channel subunit α 1D (1/100; ACC-005, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.1 (CACNA1A) antibody voltage-dependent P/Q-type calcium channel subunit α 1A (1/100; ACC-001, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.2 (CACNA1B) antibody voltage-dependent N-type calcium channel subunit α 1B (1/100; ACC1-002, Alomone Labs, Jerusalem, Israel), and anti-mouse syntaxin (1/1000, S066, Sigma, St Louis, MO, USA).

Techniques: Immunofluorescence, Labeling, Fluorescence