SNAP-25 is a novel effector of heterotrimeric G proteins modulating the strength of neurotransmission

Heterotrimeric G proteins modulate synaptic transmission in the nervous system. Presynaptic inhibition mediated by G protein-coupled receptors may involve a direct interaction between Gbetagamma subunits of the G protein and the vesicle fusion machinery. The molecular target of this pathway and mechanism underlying the inhibitory effect are unknown.; Presynaptic 5-HT GPCRs signal rapidly (≤20 ms) via Gbetagamma to inhibit release of glutamate at the lamprey giant synapses. This Gbetagamma-mediated inhibition occurs downstream from voltage-gated Ca2+ channels (established molecular targets of Gbetagamma for presynaptic inhibition) since 5-HT effectively inhibits synaptic release triggered by rapid flash photolysis of caged Ca2+. To investigate whether Gbetagamma acts within the key components of the vesicle fusion machinery (SNARE proteins), I have performed presynaptic microinjections of Botulinum toxins at the lamprey giant synapses. The effect of Botulinum neurotoxins (BoNTs) on synaptic release and 5-HT-mediated inhibition was monitored in real time with paired-cell recordings. Microinjections of BoNT/B demonstrate that cleavage of synaptobrevin in unprimed vesicles leads to an eventual exhaustion of synaptic transmission but, until that point, does not prevent 5-HT inhibition. In contrast, cleavage of the C-terminal nine amino acids of SNAP-25 by BoNT/A prevents this Gbetagamma-mediated inhibition. Moreover, a peptide containing the region of SNAP-25 cleaved by BoNT/A blocks the Gbetagamma inhibitory effect. Finally, removal of the last nine amino acids of the C terminus of SNAP-25 weakens Gbetagamma interactions with SNARE complexes. Thus, the C terminus of SNAP-25, which links synaptotagmin-I to the SNARE complex, may represent a novel molecular target of Gbetagamma for presynaptic inhibition.; Utilizing real-time binding assays, I demonstrated that Ca2+-bound synaptotagmin I competes with Gbetagamma for binding to the SNARE complex and SNAP-25 monomers. Notably, high concentrations of Ca2+ or BoNT/A cleavage of the C terminus of SNAP-25 modify the outcome of this competition in favor of Syt I. This correlates with the ability of high presynaptic Ca2+ or presynaptic microinjection of BoNT/A to prevent 5-HT inhibition mediated by Gbetagamma. Thus, Gbetagamma interferes with the Ca2+-dependent Syt I-SNARE complex binding at the C terminus of SNAP-25 to mediate inhibition of synaptic transmission.