Received for publication June 28, 2007.
Revised August 17, 2007.
Accepted for publication August 22, 2007.

G interferes with Ca²⁺-dependent binding of synaptotagmin to the SNARE complex

Abstract

Presynaptic inhibitory GPCRs can decrease neurotransmission by inducing interaction of G with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Previously, we showed that this action of G requires the carboxyl terminus of SNAP25 and is downstream of the well-known inhibition of Ca²⁺ entry through voltage-gated calcium channels (VGCC). We proposed 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 Ca²⁺-sensitive manner with binding of G 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 binding, and the G-dependent inhibiton 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 (Blackmer et al., 2001). We showed that increased presynaptic Ca²⁺ suppresses the 5-HT- and G-dependent inhibition of exocytosis. We suggest that this effect may be due to Ca²⁺-dependent competition between G 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.

Key words: Gi family, G protein regulation, Exocytosis

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