SNAP25 differentially contributes to Gi/o-coupled receptor function at glutamatergic synapses in the nucleus accumbens.

The nucleus accumbens (NAc) guides reward-related motivated behavior implicated in pathological behavioral states, including addiction and depression. These behaviors depend on the precise neuromodulatory actions of Gi/o-coupled G-protein-coupled receptors (GPCRs) at glutamatergic synapses onto medium spiny projection neurons (MSNs). Previous work has shown that discrete classes of Gi/o-coupled GPCR mobilize Gßγ to inhibit vesicular neurotransmitter release via t-SNARE protein, SNAP25. However, it remains unknown which Gαi/o systems in the NAc utilize Gßγ-SNARE signaling to dampen glutamatergic transmission. Utilizing patch-clamp electrophysiology and pharmacology in a transgenic mouse line with a C-terminal three-residue deletion of SNAP25 (SNAP25Δ3) weaking the Gßγ-SNARE interaction, we surveyed a broad cohort of Gi/o-coupled GPCRs with robust inhibitory actions at glutamatergic synapses in the NAc. We find that basal presynaptic glutamate release probability is reduced in SNAP25Δ3 mice. While κ opioid, CB1, adenosine A1, group II metabotropic glutamate receptors, and histamine H3 receptors inhibit glutamatergic transmission onto MSNs independent of SNAP25, we report that SNAP25 contributes significantly to the actions of GABAB, 5-HT1B/D, and µ opioid receptors. These findings demonstrate that presynaptic Gi/o-coupled GPCRs recruit heterogenous effector mechanisms at glutamatergic synapses in the NAc, with a subset requiring SNA25-dependent Gßγ signaling.

Gßγ inhibits exocytosis via interaction with critical residues on soluble N-ethylmaleimide-sensitive factor attachment protein-25.

Spatial and temporal regulation of neurotransmitter release is a complex process accomplished by the exocytotic machinery working in tandem with numerous regulatory proteins. G-protein ßγ dimers regulate the core process of exocytosis by interacting with the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins soluble N-ethylmaleimide-sensitive factor attachment protein-25 (SNAP-25), syntaxin 1A, and synaptobrevin. Gßγ binding to ternary SNAREs overlaps with calcium-dependent binding of synaptotagmin, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gßγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gßγ binding. Peptides that bound Gßγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gßγ-SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gßγ with SNAP-25. A peptide from the C terminus, residues 193 to 206, significantly inhibited the interaction. In addition, Ala mutants of SNAP-25 residues from the C terminus of SNAP-25, as well as from the amino-terminal region decreased binding to Gß1γ1. When SNAP-25 with eight residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gßγ, but it still interacted with synaptotagmin-1 in a Ca²âº -dependent manner and reconstituted evoked exocytosis in botulinum neurotoxin E-treated neurons. However, the mutant SNAP-25 could no longer support 5-hydroxytryptamine-mediated inhibition of exocytosis.