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¹ Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany
² The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390
³ Department of Medical Physiology and Biophysics, School of Medicine, University of Seville, 41009 Seville, Spain

Address correspondence to Jakob Balslev Sørensen, Max-Planck-Institut für Biophysikalische Chemie, Abteilung Membranbiophysik, Am Fassberg 11, D-37077 Göttingen, Germany. Fax: (49) 551-201-1688; email: jsoeren{at}gwdg.de

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.

Key Words: chromaffin cell • membrane fusion • neurosecretion • capacitance measurement • amperometry

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