A Ca(v)3.2/syntaxin-1A signaling complex controls T-type channel activity and low-threshold exocytosis

Abstract

T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.

FIGURE 1.

Ca_v3.2 channel interacts with syntaxin-1A in central neurons. A, confocal images of nRT neurons permeabilized and stained for Ca_v3.2 (green) and syntaxin-1A (red). Overlaid images and colocalized pixels (in white) are shown. B, intensity correlation analysis (ICA) plots of Ca_v3.2 and syntaxin-1A staining intensities against their respective (A-a)(B-b) values performed from the ROIs indicated in the overlaid image in A. IQC = +0.31 and +0.38 for soma and neurite regions, respectively. C, co-immunoprecipitation of syntaxin-1A from rat brain homogenate with specific anti-Ca_v3.1, anti-Ca_v3.2, and anti-Ca_v3.3 antibodies.

FIGURE 2.

Syntaxin-1A modulates Ca_v3.2 channel inactivation. A, representative Ba²⁺ current traces recorded from a Ca_v3.2- (top panel) and Ca_v3.2/Stx-1A-expressing cell (bottom panel) in response to 150 ms depolarizing steps to −20 mV from a holding potential varied from −120 mV to −50 mV (a to f). B, corresponding mean normalized steady-state inactivation curves for Ca_v3.2- (filled circles) and Ca_v3.2/Stx-1A-expressing cells (open circles). C, plot of the mean shift values in the half-inactivation potential of Ca_v3.2 channel coexpressed with the different protein combinations indicated in the figure. D, mean normalized activation curve for Ca_v3.2 (filled circles) and Ca_v3.2/Stx1A-expressing cells (open circles). Inset indicates the shift values in the half-activation potential produced upon coexpression of Stx1A or Stx1A^ΔTM. Stx-1A, syntaxin-1A; BoNT/C, botulinium neurotoxin C1.

FIGURE 3.

Syntaxin-1A interacts within the carboxy-terminal domain of Ca_v3. 2. A, schematic representation of the different constructs of intracellular regions of Ca_v3.2 used. B, whole-cell Ba²⁺ currents (top panels) recorded in response to a 150 ms depolarizing step to −20 mV from a holding potential of −85 mV before (P1) and after (P2) a 5 s hyperpolarizing pulse to −130 mV in a Ca_v3.2 (left panel), Ca_v3.2/Stx-1A (middle panel), and Ca_v3.2/Stx-1A/CD4-Ca_v3.2^Cter-expressing cell (right panel) and the corresponding mean plot of the current facilitation (I_P2/I_P1) (bottom panel). Note that the hyperpolarizing pulse produces a strong current facilitation in the presence of Stx-1A, which is competitively and specifically abolished upon co-expression of the CD4-Ca_v3.2^Cter construct. C, confocal images of living COS cells showing the translocation of the EGFP-Ca_v3.2^Cter construct (green) to the plasma membrane mediated by Stx-1A. Plasma membrane was stained with rhodamine-labeled concanavalin A (ConA-Rhod, red). Overlaid images and pixel intensity profiles of crossed sections indicated by the white line are shown. Note that Stx-1A does not translocate EGFP-Ca_v3.2^{II-III linker} fusion protein. D, co-immunoprecipitation of the CD4-EGFP-Ca_v3.2^Cter fusion protein from tsA-201 cells co-transfected with Stx-1A-Myc. The upper panel shows the immunoblot of CD4-EGFP-Ca_v3.2^Cter fusion protein in the absence (−) and presence (+) of Stx-1A-Myc using an anti-GFP antibody. *, possible degradation of the CD4-EGFP-Ca_v3.2^Cter fusion protein. The lower panel shows the results of the co-immunoprecipitation of the CD4-EGFP-Ca_v3.2^Cter fusion protein with Stx-1A-Myc using an anti-Myc antibody. In the absence of Stx-1A-Myc, the antibody alone is not able to immunoprecipitate the CD4-EGFP-Ca_v3.2^Cter fusion protein.

FIGURE 4.

Ca_v3.2 channel induces voltage-dependent exocytosis. A, representative Ca²⁺ current traces recoded from MPC 9/3L-AH cells expressing Ca_v3.2 channel alone (top panel), and in combination with CD4-Ca_v3.2^Cter (middle panel) or CD4-Ca_v3.2^{II-II linker} (bottom panel) in response to a 100 ms long depolarizing step to −20 mV from a holding potential of −90 mV. These data show that T-type currents are functional in the presence of CD4-Ca_v3.2^Cter. B, corresponding mean Ca²⁺ influx normalized by the whole-cell capacitance [Q_Ca density (pC/pF)] elicited by the 100 ms long depolarizing step to −20 mV. C, capacitance traces plotted as a function of time from the same cells shown in A. D, corresponding mean exocytosis [ΔCm (fF)] normalized as a function of Ca²⁺ entry [Q_Ca density (pC/pF)].

FIGURE 5.

Expression of the CD4-EGFP-Ca_v3.2^Cter fusion protein does not prevent exocytosis induced by intracellular calcium elevation. A, representative capacitance traces plotted as a function of time recorded from MPC 9/3L-AH cells expressing EGFP (black trace) or CD4-EGFP-Ca_v3.2^Cter (light gray) in response to the superfusion of 1 μm ionomycin (indicated by the arrow). A representative capacitance trace recorded from an EGFP-expressing cell in the absence of extracellular Ca²⁺ (EGFP, 0 Ca²⁺) is also shown (dark gray). B, corresponding mean exocytosis values [ΔCm (fF)] for EGFP in the presence (black) and the absence (dark gray) of extracellular Ca²⁺, and for CD4-EGFP-Ca_v3.2^Cter-expressing cells (light gray). C, corresponding time constants (tau) of the capacitance change obtained by fitting the rising phase of the traces with a single exponential function.