The Third Transmembrane Domain of Synaptophysin Regulates Initial Fusion Pore Dynamics during Ca2+-Triggered Exocytosis in Chromaffin cells

Synaptophysin (syp) is a major secretory vesicle protein comprising four transmembrane domains (TMDs) and a large cytoplasmic C-terminus. The C-terminus of syp has been shown to regulate exocytosis, vesicle cycling, and synaptic plasticity, but the roles of its TMDs remain unclear. SNARE TMDs line initial fusion pores, and structural work along with sequence analysis suggest that TMD III of syp may play a similar role. To test this hypothesis, we expressed TMD III tryptophan mutants in chromaffin cells from mice lacking both syp and its homolog synaptogyrin, and used amperometry to evaluate fusion pores. In contrast to SNARE TMDs, tryptophan substitutions in syp TMD III had no effect on the flux through initial fusion pores. However, these mutants increased the fraction of kiss-and-run events and decreased the initial fusion pore lifetime. Thus, syp TMD III does not line the initial fusion pore, but interacts with it to influence its stability and choice of release mode.