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

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

Gβγ interferes with Ca 2+ -dependent binding of synaptotagmin to the SNARE complex

[Ca 2+ ]; Ca 2+ concentration. SNAP25; Synaptosomal-associated protein of 25,000 dalton. MIANS; 2-(4'-maleimidylanilino) naphthalene-6-sulfonic acid. BoNT/A; botulinum toxin A. Synaptotagmin, Syt I (at MOL #39446 3 Abstract Presynaptic inhibitory GPCRs can decrease neurotransmission by inducing interaction of Gβγ with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Previously, we showed that this action of Gβγ requires the carboxyl terminus of SNAP25 and is downstream of the well-known inhibition of Ca 2+ entry through voltage-gated calcium channels (VGCC). We proposed 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 Ca 2+ -sensitive manner with binding of Gβγ to SNAP25, syntaxin1A and the assembled SNARE complex. We predict based on these findings that at high intracellular Ca 2+ concentrations, Ca 2+ -synaptotagmin I can displace Gβγ binding, and the Gβγ-dependent inhibiton 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 neurotransmissio