title: G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma.
abstract: The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma 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 Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation 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 betagamma 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 Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma 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 betagamma 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.
authors.affiliation: Department of Pharmacology & Therapeutics, Neuroscience Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: S E
authors.initials: SE
authors.lastname: Jarvis
authors.forename: J M
authors.initials: JM
authors.lastname: Magga
authors.forename: A M
authors.initials: AM
authors.lastname: Beedle
authors.forename: J E
authors.initials: JE
authors.lastname: Braun
authors.forename: G W
authors.initials: GW
authors.lastname: Zamponi
mesh_terms: D001905:Botulinum Toxins / Q000494:pharmacology; D020864:Calcium Channels, N-Type / Q000378:metabolism*; D002460:Cell Line; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins; D006801:Humans; D007668:Kidney; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050827:Syntaxin 1; D014162:Transfection
chemical_list: D020864:Calcium Channels, N-Type; C118981:G-protein Beta gamma; D044387:GTP-Binding Protein beta Subunits; D044388:GTP-Binding Protein gamma Subunits; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050765:Qa-SNARE Proteins; D050683:R-SNARE Proteins; D011993:Recombinant Fusion Proteins; C494212:STX1A protein, human; D050827:Syntaxin 1; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020962:Heterotrimeric GTP-Binding Proteins; C096324:botulinum toxin type C