G␤␥ Interferes with Ca 2ϩ -Dependent Binding of Synaptotagmin to the Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor (SNARE) Complex

Abstract

ABSTRACT Presynaptic inhibitory G protein-coupled receptors (GPCRs) can decrease neurotransmission by inducing interaction of G␤␥ with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. We have shown that this action of G␤␥ requires the carboxyl terminus of the 25-kDa synaptosomeassociated protein (SNAP25) and is downstream of the well known inhibition of Ca 2ϩ entry through voltage-gated calcium channels. We propose a mechanism in which G␤␥ and synaptotagmin compete for binding to the SNARE complex. Here, we characterized the G␤␥ interaction sites on syntaxin1A and SNAP25 and demonstrated an overlap of the G␤␥-and synaptotagmin I -binding regions on each member of the SNARE complex. Synaptotagmin competes in a Ca 2ϩ -sensitive manner with binding of G␤␥ to SNAP25, syntaxin1A, and the assembled SNARE complex. We predict, based on these findings, that at high intracellular Ca 2ϩ concentrations, Ca 2ϩ -synaptotagmin I can displace G␤␥ binding and the G␤␥-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␤␥ to inhibit neurotransmission . We showed that increased presynaptic Ca 2ϩ suppresses the 5-HT-and G␤␥-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca 2