Synaptotagmin–Ca²⁺ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation

Synaptotagmin I (Syt I), the putative Ca(2+) sensor in regulated exocytosis, has two Ca(2+)-binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca(2+)-dependent fashion. The role of Ca(2+) binding to these domains remains unclear, as efforts to address questions about Ca(2+)-triggered effector interactions have led to conflicting results. We have studied the effects of Ca(2+) on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyse the kinetics of fusion pore transitions. Elevating [Ca(2+)] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca(2+) during the actual fusion process. Analysing the Ca(2+) dependence of the fusion pore lifetime, together with the frequency of pore openings and the proportion of openings that close without dilating (kiss-and-run events) enabled us to resolve exocytosis into a sequence of kinetic steps representing functional transitions in the fusion pore. Fusion pore opening and dilation were both accelerated by Ca(2+), indicating separate Ca(2+) control over each of these steps. Ca(2+) ligand mutations in either the C2A or C2B domains of Syt I reduced fusion pore opening, but had opposite actions on the rate of fusion pore closure. These studies resolve two separate and distinct Ca(2+)-triggered steps during regulated exocytosis. The C2A and C2B domains of Syt I have different actions during these steps, and these actions may be linked to their distinctive effector interactions.