The phosphoprotein synapsin ia regulates the kinetics of dense-core vesicle release

Common fusion machinery mediates the Ca²¹-dependent exocytosis of synaptic vesicles (SVs) and dense-core vesicles (DCVs). Previously, Synapsin Ia (Syn Ia) was found to localize to SVs, essential for mobilizing SVs to the plasma membrane through phosphorylation. However, whether (or how) the phosphoprotein Syn Ia plays a role in regulating DCV exocytosis remains unknown. To answer these questions, we measured the dynamics of DCV exocytosis by using single-vesicle amperometry in PC12 cells (derived from the pheochromocytoma of rats of unknown sex) overexpressing wild-type or phosphodeficient Syn Ia. We found that overexpression of phosphodeficient Syn Ia decreased the DCV secretion rate, specifically via residues previously shown to undergo calmodulin-dependent kinase (CaMK)-mediated phosphorylation (S9, S566, and S603). Moreover, the fusion pore kinetics during DCV exocytosis were found to be differentially regulated by Syn Ia and two phosphodeficient Syn Ia mutants (Syn Ia-S62A and Syn Ia-S9,566,603A). Kinetic analysis suggested that Syn Ia may regulate the closure and dilation of DCV fusion pores via these sites, implying the potential interactions of Syn Ia with certain DCV proteins involved in the regulation of fusion pore dynamics. Furthermore, we predicted the interaction of Syn Ia with several DCV proteins, including Synaptophysin (Syp) and soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. By immunoprecipitation, we found that Syn Ia interacted with Syp via phosphorylation. Moreover, a proximity ligation assay (PLA) confirmed their phosphorylation-dependent, in situ interaction on DCVs. Together, these findings reveal a phosphorylation-mediated regulation of DCV exocytosis by Syn Ia.