Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca²⁺

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 K_D 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 K_D 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.