Gβγ interferes with Ca2+-dependent binding of synaptotagmin to the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex

Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of G beta gamma with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of G beta gamma 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 G beta gamma and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the G beta gamma interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the G beta gamma- and synaptotagmin I-binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca2+-sensitive manner with binding of G beta gamma 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 G beta gamma binding and the G beta gamma-dependent inhibition of exocytosis can be blocked. We tested this hypothesis in giant synapses of the lamprey spinal cord, where 5-HT works via G beta gamma to inhibit neurotransmission (Blackmer et al., 2001). We showed that increased presynaptic Ca2+ suppresses the 5-HT- and G beta gamma-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca2+-dependent competition between G beta gamma 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 G(i)-coupled GPCRs.