Examining synaptotagmin 1 function in dense core vesicle exocytosis under direct control of Ca2

Abstract

We tested the long-standing hypothesis that synaptotagmin 1 is the Ca2 +_ sensor for fast neurosecretion by analyzing the intracellular Ca 2+ 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 Ca 2 + - 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 Ca 2 + -threshold for secretion due to a twofold increase in the apparent K D of the Ca 2 + sensor for fast exocytosis. Single amperometric fusion events were unchanged. We conclude that Ca 2 + -dependent phospholipid binding to synaptotagmin 1 mirrors the intracellular Ca 2 + dependence of exocytosis.