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PLoS One
2007 Dec 05;212:e1273. doi: 10.1371/journal.pone.0001273.
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Depolarization-evoked secretion requires two vicinal transmembrane cysteines of syntaxin 1A.
Cohen R
,
Marom M
,
Atlas D
.
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The interactions of the voltage-gated Ca(2+) channel (VGCC) with syntaxin 1A (Sx 1A), Synaptosome-associated protein of 25 kD (SNAP-25), and synaptotagmin, couple electrical excitation to evoked secretion. Two vicinal Cys residues, Cys 271 and Cys 272 in the Sx 1A transmembrane domain, are highly conserved and participate in modulating channel kinetics. Each of the Sx1A Cys mutants, differently modify the kinetics of Cav1.2, and neuronal Cav2.2 calcium channel.We examined the effects of various Sx1A Cys mutants and the syntaxin isoforms 2, 3, and 4 each of which lack vicinal Cys residues, on evoked secretion, monitoring capacitance transients in a functional release assay. Membrane capacitance in Xenopus oocytes co-expressing Cav1.2, Sx1A, SNAP-25 and synaptotagmin, which is Bot C- and Bot A-sensitive, was elicited by a double 500 ms depolarizing pulse to 0 mV. The evoked-release was obliterated when a single Cys Sx1A mutant or either one of the Sx isoforms were substituted for Sx 1A, demonstrating the essential role of vicinal Cys residues in the depolarization mediated process. Protein expression and confocal imaging established the level of the mutated proteins in the cell and their targeting to the plasma membrane.We propose a model whereby the two adjacent transmembranal Cys residues of Sx 1A, lash two calcium channels. Consistent with the necessity of a minimal fusion complex termed the excitosome, each Sx1A is in a complex with SNAP-25, Syt1, and the Ca(2+) channel. A Hill coefficient >2 imply that at least three excitosome complexes are required for generating a secreting hetero-oligomer protein complex. This working model suggests that a fusion pore that opens during membrane depolarization could be lined by alternating transmembrane segments of Sx1A and VGCC. The functional coupling of distinct amino acids of Sx 1A with VGCC appears to be essential for depolarization-evoked secretion.
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18060067
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Figure 1. Mutation of Cys residues within the syntaxin 1A TMD disrupts depolarization-evoked capacitance transient.(A) Upper protocol, depolarizing voltage command consisting of depolarization from a holding potential of −80 mV to 0 mV for 2×500 ms, separated by 100 ms at −80 mV. Continuous monitoring of membrane capacitance of exemplary recordings, showing the effect of depolarization on membrane capacitance (Cm) in an oocyte expressing, Lc-type Ca2+ channel (Cav1.2) subunits α11.2, β2A, α2δ without (left) and with SNARE's: Sx1A 1A, SNAP-25 and synaptotagmin (right). The SNAREs and synaptotagmin were injected 24 hr after the injection of the channel subunits. (B). Monitoring Cm in oocytes expressing different Sx 1A mutants- Oocytes expressing heterologously Cav1.2 subunits (α11.2, β2A, α2δ), SNAP-25, synaptotagmin I with either one of the Sx mutants: C271V/C272V, C272V, C271V, or V271C/V272C (Trus et al., 2001; Arien et al., 2003) or a truncated Sx 1A (1–167) (C) Summary: effect of depolarization on Cm. Groups as in (B). ΔCm, depolarization-induced change of membrane capacitance; bars show mean±SEM (n = 13). Inset, The effect of depolarization on membrane current (InA mean±SEM, n = 13–18). (D) Monitoring differences in Cm induced by different pulse duration via excitosome composed of Sx 1A and Sx CC/VV- Capacitance induced by varying depolarizing pulses as indicated; bars show mean±SEM (n = 13–15).
Figure 2. Expression and interaction of RFP-Sx1A and RFP-Sx1A(CC/VV) with Cav1.2.Superimposed current traces of GFP-α11.2/β2A/α2/δ (5/5/5 ng/oocyte) co expressed with (A) RFP-Sx1A (0.8 ng/oocyte and 1.6 ng/oocyte) or (B) RFP-Sx1A(CC/VV) (0.8 and 1.6 ng/oocyte) in 10 mM Ba2+. Currents were elicited from a holding potential of –80 mV to +10 mV in response to 500 ms pulse (upper panels). Leak-subtracted peak current–voltage relations collected data from oocytes expressing GFP-α11.2/β2A/α2/δ (5/5/5 ng/oocyte) without (-○-) and with RFP-Sx1A (0.8 ng/oocyte -•- and 1.6 ng/oocyte -▪-) (A) and RFP-Sx1A(CC/VV). Currents were elicited in response to 500 ms pulse from a holding potential of –80 mV to various test potentials at 5-sec intervals (B) (middle). Activation rate constants (τ, mean±SEM, n = 12) of currents generated in oocytes by GFP-α11.2/β2A/α2/δ without (-○-) and with RFP-Sx1A (0.8 ng/oocyte -•- and 1.6 ng/oocyte -▪-) (A) and RFP-Sx1A(CC/VV) (B) (lower). The data points correspond to the mean±SEM of currents (n = 8-14) at each experimental point. Two-sample Student's t tests assuming unequal variance were applied, and P values <0.01 were obtained (C) Dose dependent of GFP-α11.2/β2A/α2/δ current inhibition, plotted against increasing RFP-Sx1A and RFP-Sx1A(CC/VV) RNA concentration injected into oocytes. (D) Sx1A expression was tested in a western blot analysis of oocyte plasma membrane fraction co expressing GFP-α11.2/β2A/α2/δ and 1.6 ng/oocyte RFP-Sx1A, 1.6 ng/oocyte RFP-Sx1A(CC/VV), 1.6 ng/oocyte Sx1A. Proteins were detected with anti-Sx1A antibodies.
Figure 3. Expression and localization of Sx1A and Sx1A(CC/VV) on the cell membrane.(A) Western blot analysis of membrane fraction of oocyte expressing GFP-α11.2/β2A/α2/δ (5/5/5 ng/oocyte) with either RFP-Sx1A (1.6 ng/oocyte) or RFP-Sx1A (CC/VV) (1.6 ng/oocyte) or Sx1A (1.6 ng/oocyte), with or without SNAP-25 (1.6 ng/oocyte) and Syt I (3.2 ng/oocyte) (Excitosome, Ex), using anti-Sx1Aa antibodies. (B) Oocytes were injected with cRNA mixture encoding the excitosome complex (GFP-Cav1.2/RFP-Sx1A/SNAP-25/SytI) or (C) (GFP-Cav1.2/RFP-Sx1A(CC/VV)/SNAP-25/SytI) and fluorescence was measured using confocal microscopy. GFP-Cav1.2 fluorescence (upper panel) and RFP-Sx1A fluorescence (middle panel) were localized at the cell membrane and a merge of the showed co-localization of both proteins. The enlarged area is depicted at the right hand side. The experiment was repeated two times with 4 oocytes in each group.
Figure 4. Phenyl arsene oxide abolished depolarization evoked ΔCm.(A) Continuous monitoring of membrane capacitance induced by a depolarizing voltage command from a holding potential of −80 mV to 0 mV of 2×500 ms, separated by 100 ms at −80 mV in an oocyte expressing, Cav1.2 subunits, α11.2, β2A, α2δ without (upper left) and with SNARE's: Sx1A, SNAP-25 and SytI (upper right), and with either PAO (10 µM) (lower left) or PAO (10 µm) followed by 2 mM BAL (lower right). (B) Summary of effect of depolarization on Cm. Groups as in the exemplary recordings shown in (A). ΔCm, depolarization-induced change of membrane capacitance; (left) bars show mean±SEM (n = 13) and the Effect of depolarization on mean peak Ba2+ currents: InA (mean±SEM, n = 11; right).
Figure 5. Modulation of Cav1.2 kinetics by syntaxin isoforms.Oocytes were injected with α11.2 (2 ng/oocyte), β2A (5 ng/oocyte), α2δ (5 ng/oocyte) and 24 hr later, with either one of the syntaxin isoforms (2 ng/oocyte). (A) At day 6 after cRNA injection Ba2+ currents were elicited from a holding potential of –80 mV by voltage steps applied at 5-sec intervals to test potentials between –35 to +45 mV in response to 160 ms pulse duration. Representative traces of inward currents are shown. (B) Leak subtracted peak-current relationship: collected data form oocytes expressing Cav1.2 (o) and Cav1.2 with each one of the Sx isoforms (•). The data points correspond to the mean±SEM of currents (n = 8) at each experimental point. (C) The effect of syntaxin isoforms on I/Imax ratio. Peak current amplitudes normalized to maximum current (I/Imax) (data from B) are plotted against test potentials and displayed with a Boltzmann fit (mean±S.E.M; n = 8–10; more details in Experimental Procedures). (D) The averaged time constant of activation (τact mean±S.E, n = 11–13) are plotted against test pulses between −25 and +20 mV in the absence of (o) and in the presence (•) of Sx isoform as indicated.
Figure 6. Comparison of syntaxin isoforms effects on the kinetics of Cav1.2. Cav1.2 subunits were co-injected with syntaxin isoforms (data from Figure 3).Ba2+ currents were elicited from a holding potential of –80 mV by a voltage step applied to +4 mV in response to 160 ms pulse duration. (A) Superposition of representative online leak-subtracted current traces measured with Sx isoforms as indicated with voltage protocols diagramed at the top. (B) The first 30 ms of the response is shown. The normalized traces show a shift of Cav1.2 activation by Sx isoforms. (C) Normalized conductance–voltage (G/Gmax) relationship obtained from (Fig. 3.B) displayed with a Boltzmann fit. The mid-point of activation (V1/2) and the Boltzmann slope (k) of Cav1.2 were: V1/2 = −7.6±0.2 mV, k = 6.3±0.3; with Sx 1A, V1/2 = −3.5±1.9 mV; k = 5.9±1; with Sx 2, V1/2 = −1.8±1.9 mV; k = 5±0.6±1; with Sx3, V1/2 = −3.7±0.9 mV, k = 5.9±0.5; and with Sx 4, V1/2 = −3.5±1.4 mV; k = 6.3±0.7. (D) Peak current amplitudes normalized to maximum current (I/Imax) (data from Fig. 3B) are plotted against test potentials and displayed with a Boltzmann fit. The data points correspond to the mean±S.E.M. (n = 10–12). Statistical significance was determined by Student's t-test.
Figure 7. Assembly of the excitosome with syntaxin isoforms does not support depolarization-induced secretion.(A) Oocytes were injected with Cav1.2 subunits (as detailed in legend to Fig. 1) and 24 hr later with cRNA encoding SNAP-25, Syt 1 and either one of the syntaxin isoforms. Capacitance steps were elicited by two consecutive pulses of 500 ms, 100 ms apart as depicted in the protocol in Fig. 1D. Monitoring Cm in representative oocytes expressing heterologously Cav1.2 without and with SNAP-25, synaptotagmin and different syntaxin 1A isoforms. The amino acid sequence of Sx isoforms TMD are shown (left). (B) Summary of the exemplary recordings shown in (A) of the Sx isoforms effect on Cm induced by membrane depolarization of Cav1.2 0.71±0.07 nF; n = 11; and with: Sx 1A, 2.65±0.17 nF (n = 20), Sx 2, 1.04±0.13 nF (n = 16), Sx 3, 0.86±0.12 nF (n = 12) and Sx 4, 0.48±0.11 (n = 12) (Groups as in A). insert, Average the corresponding peak currents amplitudes of VGCC expressed with SNAP-25, synaptotagmin and syntaxin isoforms, as indicated.
Figure 8. Assembly of VGCC Sx 1A, SNAP-25, and SytI, to generate a releasing complex; A Schematic model.The voltage -gated Ca2+ channel is schematically illustrated as a transmembrane barrel (yellow), and Sx 1A as a single transmembrane domain (red). (A) The Ca2+ channel, interact with Sx 1A transmembrane domain either via Cys 271 or Cys 272 residues, where one channel molecule interacts with one Sx1A (left). A simultaneous interaction of one Sx 1A molecule with two adjacent VGCC molecules via two vicinal Cys resides, lead to the Sx1A lashing two VGCC molecules, consequently, three Sx1A together with three VGCC molecules generate a secreting competent cluster (right). For simplicity, SNAP-25, and synaptotagmin were not inserted. (B) Top view of the cluster formed by three Sx1A and three Ca2+ channel molecules, clearly illustrates the fusion pore that traverses the plasma membrane to form a circle (shaded area).
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