Molecular Determinants of Syntaxin 1 Modulation of N-type Calcium Channels

Abstract

We have previously reported that syntaxin 1A, a component of the presynaptic SNARE complex, directly modulates N-type calcium channel gating in addition to promoting tonic G-protein inhibition of the channels, whereas syntaxin 1B affects channel gating but does not support G-protein modulation (Jarvis, S. E., and Zamponi, G. W. (2001) J. Neurosci. 21, 2939 -2948). Here, we have investigated the molecular determinants that govern the action of syntaxin 1 isoforms on N-type calcium channel function. In vitro evidence shows that both syntaxin 1 isoforms physically interact with the G-protein ␤ subunit and the synaptic protein interaction (synprint) site contained within the N-type calcium channel domain II-III linker region. Moreover, in vitro evidence suggests that distinct domains of syntaxin participate in each interaction, with the COOH-terminal SNARE domain (residues 183-230) binding to G␤ and the N-terminal (residues 1-69) binding to the synprint motif of the channel. Electrophysiological analysis of chimeric syntaxin 1A/1B constructs reveals that the variable NH 2 -terminal domains of syntaxin 1 are responsible for the differential effects of syntaxin 1A and 1B on N-type calcium channel function. Because syntaxin 1 exists in both "open" and "closed" conformations during exocytosis, we produced a constitutively open form of syntaxin 1A and found that it still promoted G-protein inhibition of the channels, but it did not affect N-type channel availability. This state dependence of the ability of syntaxin 1 to mediate N-type calcium channel availability suggests that syntaxin 1 dynamically regulates N-type channel function during various steps of exocytosis. Finally, syntaxin 1A appeared to compete with G␥ for the G␤ subunit both in vitro and under physiological conditions, suggesting that syntaxin 1A may contain a G-protein ␥ subunit-like domain.Calcium influx through N-type calcium channels is a key step in neurotransmitter release from presynaptic nerve termini (1, 2). In mammalian neurons, these channels physically associate with proteins of the presynaptic vesicle docking and release machinery, notably syntaxin 1, soluble NSF attachment protein (SNAP) 1 25, synaptotagmin, cysteine string protein, Rab3-interacting molecules (RIMs), and RIM-binding protein 2, thus localizing synaptic vesicles close to the source of extracellular calcium (3-12). The interaction of SNAP25 and syntaxin 1A or 1B with the N-type calcium channel acts as a negative feedback mechanism, such that N-type calcium channel availability is reduced in the presence of these proteins, which is reflected as a negative shift in the half-inactivation potential of the channels (13-16). We have recently shown that this effect is abolished when both syntaxin 1 and SNAP25 are present concomitantly, or following coexpression of nSec-1 (also called , and that protein kinase C-dependent phosphorylation of the channel abolishes the shift in steady state inactivation (16). Together, these findings indicate that the effects of syntaxin 1 on channel avail...

Old proteins, developing roles: The regulation of calcium channels by synaptic proteins

“…Syntaxin 1A (but not 1B) also mediates a second inhibitory action on N-type calcium channels by inducing a tonic G protein inhibition of N-type channels 61,67,69,71,72 which may involve a syntaxin 1A dependent colocalization of the channel with Gβγ subunits. Unlike the effect of syntaxin 1A on channel availability, this modulation persists in the presence of SNAP-25.…”

“…17,60 It may also serve as an auto-inhibitory domain. 61 In addition to anchoring to the synprint site, another region of the H3 domain binds elsewhere on the channel to regulate its biophysical properties. 21 Neuronal SNAP-25 and its ubiquitously expressed homolog SNAP-23 are members of a family of synaptosome-associated proteins that interact with syntaxin 1A to form the t-SNARE complex.…”

“…97 nSec1 binds with high affinity to syntaxin 1A and to the SNARE complex, 98 stabilizing its closed conformation which antagonizes priming 96,[99][100][101] by preventing binding to SNARE partners and as mentioned earlier, syntaxin 1A-mediated inhibition of N-type channels. 61,67 nSec1 is a critical regulator of the exocytotic machinery with null mutants exhibiting no neurotransmission. 100 Recent studies suggest that nSec1 may be the key regulator of the machinery, controlling every step of the exocytotic pathway and dictating how closely SNAREs interact.…”

Expression and Modulation of an Invertebrate Presynaptic Calcium Channel α1 Subunit Homolog

“…Syntaxin1 Regulation of the LCa v 2a-5ЈrbA Channel-We have shown previously that unlike the rat N-type calcium channel (1,2,26,28,32,50), the LCa v 2a calcium channel is incapable of directly interacting with syntaxin1 (21). This is due primarily to the absence of the synaptic protein interaction site in the LCa v 2a II-III linker region (Fig.…”

A Plasma Membrane Syntaxin Is Phosphorylated in Response to the Bacterial Elicitor Flagellin

“…For N-type calcium channels, direct inhibitory interactions with human syntaxin 1 have been established (43), whereas for the cystic fibrosis transmembrane conductance regulator channel, both direct interaction and regulation of cell surface concentration via exocytosis are being discussed (44). In plant cells, elicitors induce rapid changes of ion fluxes in plant cells, and it is conceivable that AtSyp122 plays a role in modulating these fluxes.…”