Abstract
Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of Gβγ with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of Gβγ requires the carboxyl terminus of the 25-kDa synaptosome-associated protein (SNAP25) and is downstream of the well known inhibition of Ca2+ entry through voltage-gated calcium channels. We propose a mechanism in which Gβγ and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the Gβγ interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the Gβγ- and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-sensitive manner with binding of Gβγ to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca2+ concentrations, Ca2+-synaptotagmin I can displace Gβγ binding and the Gβγ-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via Gβγ to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and Gβγ-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between Gβγ and synaptotagmin I for SNARE binding. This type of dynamic regulation may represent a novel mechanism for modifying transmitter release in a graded manner based on the history of action potentials that increase intracellular Ca2+ concentrations and of inhibitory signals through Gi-coupled GPCRs.
Footnotes
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This work was supported by National Institutes of Health grants EY10291 (to H.E.H.) and MH64763 (to S.A.) and by an American Heart Associate predoctoral fellowship (to E.J.Y.).
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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doi:10.1124/mol.107.039446.
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ABBREVIATIONS: GPCR, G protein-coupled receptor; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; t-SNARE, target membrane-associated snare; SNAP25, 25-kDa synaptosome-associated protein; DTT, dithiothreitol; OG, n-octyl β-d-glucopyranoside; PAGE, polyacrylamide gel electrophoresis; GST, glutathione transferase; MIANS, 2-(4′-maleimidylanilino) naphthalene-6-sulfonic acid; MOPS, 3-(N-morpholino)propanesulfonic acid; BoNT/A, botulinum toxin A; EPSC, excitatory postsynaptic current; 5-HT, 5-hydroxytryptamine.
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received June 27, 2007.
- Accepted August 22, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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