G Protein Modulation of N-type Calcium Channels Is Facilitated by Physical Interactions between Syntaxin 1A and Gβγ

Abstract

The direct modulation of N-type calcium channels by G protein ␤␥ subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of Ntype channels and G␤␥ have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by G␤␥. When syntaxin 1A was coexpressed with (N-type) ␣ 1B ؉ ␣ 2 -␦ ؉ ␤ 1b channels in tsA-201 cells, the channels underwent a 18 mV negative shift in halfinactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein ␤␥ subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that G␤␥ physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with G␤␥ and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein ␤␥ subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.

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

“…22 Coexpression of syntaxin 1A (or 1B) with N-type or P/Q-type channels results in inhibition of channel activity, by virtue of a strong hyperpolarizing shift in the voltage dependence of steady state inactivation of the channel. 21,[67][68][69] A similar effect is observed upon co expression of N-type channels with SNAP-25. 67 For N-type channels, the inhibitory effects of syntaxin and SNAP-25 are reversed when both proteins are co expressed with the channel.…”

“…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.…”

RGS12 Interacts with the SNARE-binding Region of the Cav2.2 Calcium Channel

“…Binding of syntaxin to this channel region plays a role in voltage-dependent inhibition (23) and is thought to stabilize the binding of G protein ␤␥ subunits to the channel (24). Synaptotagmin also binds to the channel in this region upon depolarization in a calcium-dependent manner (25).…”

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

“…1B). Hence, one would predict that the coexpression of syntaxin1, which in rat N-type calcium channels results in a pronounced hyperpolarizing shift in half-inactivation potential (28,32,50), should not affect LCa v 2a-5ЈrbA channel gating. Rather than using rat syntaxin1A or 1B, which we have characterized previously, we used Lymnaea syntaxin1 (Lsytx 1 ) for coexpression studies.…”

“…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.…”