We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2+ sensor for fast neurosecretion by analyzing the intracellular Ca2+ dependence of large dense-core vesicle exocytosis in a mouse strain carrying a mutated synaptotagmin C2A domain. The mutation (R233Q) causes a twofold increase in the KD of Ca2+-dependent phospholipid binding to the double C2A-C2B domain of synaptotagmin. Using photolysis of caged calcium and capacitance measurements we found that secretion from mutant cells had lower secretory rates, longer secretory delays, and a higher intracellular Ca2+-threshold for secretion due to a twofold increase in the apparent KD of the Ca2+ sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca2+-dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca2+ dependence of exocytosis.
Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2+
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Jakob B. Sørensen, Rafael Fernández-Chacón, Thomas C. Südhof, Erwin Neher; Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2+ . J Gen Physiol 1 September 2003; 122 (3): 265–276. doi: https://doi.org/10.1085/jgp.200308855
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