Synaptophysin (syp) is a major protein of secretory vesicles with four transmembrane domains (TMDs) and a large cytoplasmic C-terminus. Syp has been shown to regulate exocytosis, vesicle cycling, and synaptic plasticity through its C-terminus. However, the roles of its TMDs remain unclear. The TMDs of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are thought to line initial fusion pores, and structural work together with sequence analysis suggest that TMD III of syp may play a similar role. To test this hypothesis, we performed tryptophan scanning experiments of TMD III in chromaffin cells 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, a number of these mutants increased the fraction of kiss-and-run events and decreased the initial fusion pore lifetime. These results indicate that TMD III stabilizes the initial fusion pore and controls the initial choice between kiss and run and full fusion. Late-stage fusion pores were not impacted by TMD III mutations. These results indicate that syp TMD III does not line the initial fusion pore. However, its impact on pore dynamics suggests that it interacts with a SNARE protein implicated as a part of the fusion pore that forms at the onset of exocytosis.

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