Synaptotagmin–Ca2+ triggers two sequential steps in regulated exocytosis in rat PC12 cells: fusion pore opening and fusion pore dilation
Journal
Journal of Physiology
Journal Volume
570
Journal Issue
2
Pages
295
Date Issued
2006-01-01
Author(s)
Abstract
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. © 2006 The Authors. Journal compilation © 2006 The Physiological Society.
Publisher
WILEY
Type
journal article
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