Gβγ directly modulates vesicle fusion by competing with synaptotagmin for binding to neuronal SNARE proteins embedded in membranes

G_i/o-coupled G protein-coupled receptors can inhibit neurotransmitter release at synapses via multiple mechanisms. In addition to G-mediated modulation of voltage-gated calcium channels (VGCC), inhibition can also be mediated through the direct interaction of G subunits with the soluble N-ethylma-leimide attachment protein receptor (SNARE) complex of the vesicle fusion apparatus. Binding studies with soluble SNARE complexes have shown that G binds to both ternary SNARE complexes, t-SNARE heterodimers, and monomeric SNAREs, competing with synaptotagmin 1(syt1) for binding sites on t-SNARE. However, in secretory cells, G, SNAREs, and synaptotagmin interact in the lipid environment of a vesicle at the plasma membrane. To approximate this environment, we show that fluorescently labeled G interacts specifically with lipid-embedded t-SNAREs consisting of full-length syntaxin 1 and SNAP-25B at the membrane, as measured by fluorescence polarization. Fluorescently labeled syt1 undergoes competition with G for SNARE-binding sites in lipid environments. Mutant G subunits that were previously shown to be more efficacious at inhibiting Ca²-triggered exocytotic release than wild-type G were also shown to bind SNAREs at a higher affinity than wild type in a lipid environment. These mutant G subunits were unable to inhibit VGCC currents. Specific peptides corresponding to regions on G and G shown to be important for the interaction disrupt the interaction in a concentration-dependent manner. In in vitro fusion assays using full-length t- and v-SNAREs embedded in liposomes, G inhibited Ca²/synaptotagmin-dependent fusion. Together, these studies demonstrate the importance of these regions for the G-SNARE interaction and show that the target of G, downstream of VGCC, is the membrane-embedded SNARE complex.