Syntaxin Modulation of Slow Inactivation of N-Type Calcium Channels

Abstract

Syntaxin, a membrane protein vital in triggering vesicle fusion, interacts with voltage-gated N- and P/Q-type Ca(2+) channels. This biochemical association is proposed to colocalize Ca(2+) channels and presynaptic release sites, thus supporting rapid and efficient initiation of neurotransmitter release. The syntaxin channel interaction may also support a novel signaling function, to modulate Ca(2+) channels according to the state of the associated release machinery (Bezprozvanny et al., 1995; Wiser et al., 1996; see also Mastrogiacomo et al., 1994). Here we report that syntaxin 1A (syn1A) coexpressed with N-type channels in Xenopus oocytes greatly promoted slow inactivation gating, but had little or no effect on the onset of and recovery from fast inactivation. Accordingly, the effectiveness of syntaxin depended strongly on voltage protocol. Slow inactivation was found for N-type channels even in the absence of syntaxin and could be distinguished from fast inactivation on the basis of its slow kinetics, distinct voltage dependence (voltage-independent at potentials higher than the level of half-inactivation), and temperature independence (Q(10), approximately 0.8). Trains of action potential-like stimuli were more effective than steady depolarizations in stabilizing the slowly inactivated condition. Agents that stimulate protein kinase C decreased the inhibitory effect of syntaxin on N-type channels. Application of BoNtC1 to cleave syntaxin sharply attenuated the modulatory effects on Ca(2+) channel gating, consistent with structural analysis of syntaxin modulation, supporting use of this toxin to test for the impact of syntaxin on Ca(2+) influx in nerve terminals.

Functional Interaction of Auxiliary Subunits and Synaptic Proteins With CaV1.3 May Impart Hair Cell Ca2+Current Properties

“…Ca 2ϩ channels at presynaptic terminals may be located as close as approximately 10 nm from the docking site for a synaptic vesicle, and there is evidence of physical and functional interaction between the channels and synaptic proteins (Atlas 2001;Degtiar et al 2000;Stanley and Mirotznik 1997). The Ca V 1.3␣ 1 channel is the predominant presynaptic Ca 2ϩ channel that mediates transmitter release in hair cells.…”

“…Botulinum toxin C is an endopeptidase neurotoxin that cleaves syntaxin 1A, disrupting its interaction with Ca 2ϩ channels and inhibiting neurotransmitter release (Degtiar et al 2000;Rettig et al 1996;Sheng et al 1994). To test whether the effects of syntaxin on Ca V 1.3␣ 1 current in HEK 293 cells also occur in hair cells, we directly examined the role played by botulinum toxin C on hair cell Ca 2ϩ currents.…”

“…The DHP-sensitive Ca 2ϩ channel in hair cells has been cloned from the chick basilar papilla and identified as ␣ 1D (Kollmar et al 1997a) or Ca V 1.3 ␣ 1 channel (Ertel et al 2000). In contrast to other Ca 2ϩ channels, wherein the heterologous expression of the pore-forming ␣ subunit closely mirrors the native channel current, the biophysical properties of the expressed Ca V 1.3 are quite distinct from typical hair cell Ca 2ϩ currents (Bell et al 2001;Koschak et al 2001;Mikami et al 1989;Seino et al 1992;Williams et al 1992;Xu and Lipscombe 2001).…”

Cumulative inactivation of N-type CaV2.2 calcium channels modified by alternative splicing

“…Only in the case of Ca V 3 channels that deactivate relatively slowly following action potential repolarization has open-state inactivation been implicated in cumulative inactivation (9)(10)(11). Several factors modulate the susceptibility of voltage-gated calcium channels to cumulative inactivation, including channel subtype (6), G protein activation (3), interaction with presynaptic proteins (12,13), and alternative splicing (10,11). However, a limited number of studies have investigated the mechanisms by which cumulative inactivation is modulated (12).…”

“…Several factors modulate the susceptibility of voltage-gated calcium channels to cumulative inactivation, including channel subtype (6), G protein activation (3), interaction with presynaptic proteins (12,13), and alternative splicing (10,11). However, a limited number of studies have investigated the mechanisms by which cumulative inactivation is modulated (12).…”