Abstract

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 βγ dimers regulate the core process of exocytosis by interacting with the SNARE proteins SNAP-25, syntaxin 1A, and synaptobrevin. Gβγ binding to ternary SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors) overlaps with synaptotagmin’s calcium-dependent binding, inhibiting synaptotagmin-1 binding and fusion of the synaptic vesicle. To further explore the binding sites of Gβγ on SNAP-25, peptides based on the sequence of SNAP-25 were screened for Gβγ binding. Peptides that bound Gβγ were subjected to alanine scanning mutagenesis to determine their relevance to the Gβγ–SNAP-25 interaction. Peptides from this screen were tested in protein-protein interaction assays for their ability to modulate the interaction of Gβγ with SNAP-25. A peptide from the C-terminus, residues 193-206, significantly inhibited the interaction. Additionally, Ala mutants of SNAP-25 residues from the C-terminus of SNAP-25, as well as from the amino terminal region decreased binding to Gβ₁γ₁. When SNAP-25 with 8 residues mutated to alanine was assembled with syntaxin 1A, there was significantly reduced affinity of this mutated t-SNARE for Gβγ, but it still interacted with synaptotagmin-1 in a Ca²⁺-dependent manner and reconstituted evoked exocytosis in BoNT/E-treated neurons. However, the mutant SNAP-25 could no longer support 5-HT-mediated inhibition of exocytosis.