The transmembrane domain of synaptobrevin influences neurotransmitter flux through synaptic fusion pores

Abstract

The soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins synaptobrevin (Syb), syntaxin, and SNAP-25 function in Ca2+-triggered exocytosis in both endocrine cells and neurons. The transmembrane domains (TMDs) of Syb and syntaxin span the vesicle and plasma membrane, respectively, and influence flux through fusion pores in endocrine cells as well as fusion pores formed during SNARE-mediated fusion of reconstituted membranes. These results support a model for exocytosis in which SNARE TMDsform the initial fusion pore. The present study sought to test this model in synaptic terminals. Patch-clamp recordings of miniature EPSCs (mEPSCs) were used to probe fusion pore properties in cultured hippocampal neurons from mice of both sexes. Mutants harboring tryptophan at four different sites in the Syb TMD reduced the rate-of-rise of mEPSCs. A computer model that simulates glutamate diffusion and receptor activation kinetics could account for this reduction in mEPSC rise rate by slowing the flux of glutamate through synaptic fusion pores.TMDmutations introducing positive charge also reduced the mEPSC rise rate, but negatively charged residues and glycine, which should have done the opposite, had no effect. The sensitivity of mEPSCs to pharmacological blockade of receptor desensitization was enhanced by a mutation that slowed the mEPSC rate-of-rise, suggesting that the mutation prolonged the residence of glutamate in the synaptic cleft. The same four Syb TMD residues found here to influence synaptic release were found previously to influence endocrine release, leading us to propose that a similar TMD-lined fusion pore functions widely in Ca2+-triggered exocytosis in mammalian cells.