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.
Wang
Chih-Tien
CT
Department of Physiology, University of Wisconsin, Madison, 53706, USA.
Bai
Jihong
J
Chang
Payne Y
PY
Chapman
Edwin R
ER
Jackson
Meyer B
MB
D000818:Animals; D002118:Calcium / Q000502:physiology; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003627:Data Interpretation, Statistical; D005089:Exocytosis / Q000187:drug effects* / Q000502:physiology; D008024:Ligands; D008564:Membrane Potentials / Q000187:drug effects* / Q000502:physiology; D009154:Mutation; D016716:PC12 Cells; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology; D017434:Protein Structure, Tertiary / Q000502:physiology; D051381:Rats; D050863:Synaptotagmin I / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology / Q000502:physiology*
D008024:Ligands; D020033:Protein Isoforms; D050863:Synaptotagmin I; D002118:Calcium