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Received August 24, 2005
Revised October 5, 2005
Accepted after revision November 11, 2005
1 UCSD
2 Mass General Hospital
3 University of Wisconsin
4 Howard Hughes Medical Institute and University of Wisconsin
* To whom correspondence should be addressed. E-mail: mjackson{at}physiology.wisc.edu.
Synaptotagmin I (Syt I), the putative Ca2+ sensor in regulated exocytosis, has two Ca2+ binding modules, the C2A and C2B domains, and a number of putative effectors to which Syt I binds in a Ca2+-dependent fashion. The role of Ca2+ binding to these domains remains unclear as efforts to address questions about Ca2+-triggered effector interactions have lead to conflicting results. We have studied the effects of Ca2+ on fusion pores using amperometry to follow the exocytosis of single vesicles in real time and analyze the kinetics of fusion pore transitions. Elevating [Ca2+] in permeabilized cells reduced the fusion pore lifetime, indicating an action of Ca2+ during the actual fusion process. Analyzing the Ca2+-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 Ca2+, indicating separate Ca2+ control over each of these steps. Ca2+ 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 Ca2+ 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.
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