Synaptotagmin-7 Enhances Facilitation of Ca_v2.1 Calcium Channels

Direct binding of Syt-7α to the synprint site of P/Q-type Ca_v2.1 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_v2.1 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_v2.1 channel. GST-Syt-7α were immobilized on glutathione-Sepharose beads and incubated with His-Ca_v2.1 or His-Ca_v1.2 using different Ca²⁺ concentrations (10 μm to 1 mm). The binding experiments were performed in a Ca²⁺ buffering system containing: 5 mm N-hydroxyethyl ethylenediaminetriacetic, 50 mm HEPES (pH 7.2), and 150 mm NaCl. The free Ca²⁺ concentrations were estimated using the MAX CHELATOR software. The Ca²⁺ concentrations used were 10 μm, 50 μm, 100 μm, 300 μm, 500 μm, 700 μm, and 1 mm. After extensive washing, GST-Syt-7α bound to the beads were eluted with 15 mm reduced glutathione (GSH) in 50 mm Tris-HCl (pH 8) and proceed to electrophoresis and immunoblotting. The bound His-Ca_v2.1 synprint (724–981) proteins were detected with anti-His antibody. Because different Ca²⁺ 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.

Syt-7α accelerates the onset of facilitation of Ca_v2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 mm extracellular Ca²⁺ and 0.5 mm 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_v2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

Effect of Syt-7α on prepulse facilitation of Ca_v2.1 at physiological Ca²⁺ levels. Inset top, Pulse protocol. Currents recorded with 2 mm extracellular Ca²⁺ and 0.5 mm 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.

Effect of Syt-7α on the decay from facilitation of Ca_v2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca²⁺ 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 Δt = 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.

Syt-7α potentiates Ca_v2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca²⁺ current was recorded using 10 mm Ca²⁺ and 0.5 mm EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca²⁺ 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.

Effect of Syt-7α on prepulse facilitation of Ca_v2.1 channel. Facilitation of voltage-dependent activation of Ca²⁺ 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_v2.1 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_v2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

Effect of Syt-7α prepulse facilitation of Ca_v2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 mm extracellular Ca²⁺ and 0.5 mm 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.

Syt-7 isoforms α, β, and γ co-immunoprecipitate with Ca_v2.1 channels. A, Left, In transfected tsA-201 cells, co-immunoprecipitation experiments show the binding of Syt-7β and α_1A subunit of Ca_v2.1. Ca_v2.1 channels were immunoprecipitated with an anti-Ca_v2.1 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_v2.1 antibody. B, Left, In tsA-201 cells co-transfected with the α_1A subunit of Ca_v2.1 channels together with Syt-7γ, Ca_v2.1 channels were immunoprecipitated with an anti-Ca_v2.1 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_v2.1 antibody. C, Left, In tsA-201 cells co-transfected with the α_1A subunit of Ca_v2.1 channels along with Syt-7α, β, and γ, Ca_v2.1 channels were immunoprecipitated with an anti-Ca_v2.1 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_v2.1 antibody. Because different Ca²⁺ 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_v2.1 expressed alone gives similar peak Ba²⁺ and Ca²⁺ currents as Ca_v2.1 + Syt7-αβγ (Extended Data Fig. 8-1).

Syt-7β and Syt-7γ differentially modulate facilitation of Ca_v2.1 channels. Inset, Pulse protocol. Currents recorded with 10 mm extracellular Ca²⁺ and 0.5 mm 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_v2.1 channels (Extended Data Fig. 9-1).

Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca_v2.1 channels. Inset, Pulse protocol. Currents recorded with 10 mm extracellular Ca²⁺ and 0.5 mm 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_v2.1 alone. B, Ca_v2.1 with Syt-7γ. C, Overlay of panels A, B. D, V₅₀ values for results in panel C. E, Ca_v2.1 alone. F, Ca_v2.1 with Syt-7γ. G, overlay of panels D, E. H, V₅₀ 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_v2.1 channels (Extended Data Fig. 10-1).