Abstract

Gi/o-coupled G-protein coupled receptors (GPCRs) can exert an inhibitory effect on vesicle release through several G-protein driven mechanisms, more than one of which may be concurrently present in individual presynaptic terminals. The synaptosomal-associated protein of 25 kDa (SNAP25) is a key downstream effector of G protein betagamma (Gβγ) subunits. It has previously been shown that proteolytic cleavage of SNAP25 by botulinum toxin A (BoNT/A) reduces the ability of Gβγ to compete with the calcium sensor synaptotagmin 1 (Syt1) for binding to SNAP25 in a calcium-dependent manner. These truncated SNAP25 proteins sustain a low level of exocytosis but are unable to support serotonin-mediated inhibition of exocytosis in lamprey spinal neurons. Here, we generate a SNAP-5 extreme C-terminal mutant that is deficient in its ability to bind Gβγ while retaining normal calcium-dependent Syt1 binding to SNARE and vesicle release. The SNAP25Δ3 mutant, in which residue G204 is replaced by a stop codon, features a partial reduction in Gβ1γ2 binding in vitro as well as a partial reduction in the ability of the lamprey 5HT1b-type serotonin receptor to reduce excitatory postsynaptic current (EPSC) amplitudes, an effect previously shown to be mediated through the interaction of Gβγ with SNAP25. Syt1 calcium-dependent binding to SNAP25Δ3 was reduced by a small extent compared to wild-type. We conclude that the extreme C-terminus of SNAP25 is a critical region for the Gβγ-SNARE interaction.