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.
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1 September 2003
Article|
August 25 2003
Examining Synaptotagmin 1 Function in Dense Core Vesicle Exocytosis under Direct Control of Ca2+
Jakob B. Sørensen,
Jakob B. Sørensen
1Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany
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Rafael Fernández-Chacón,
Rafael Fernández-Chacón
2The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390
3Department of Medical Physiology and Biophysics, School of Medicine, University of Seville, 41009 Seville, Spain
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Thomas C. Südhof,
Thomas C. Südhof
2The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390
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Erwin Neher
Erwin Neher
1Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany
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Jakob B. Sørensen
1Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany
Rafael Fernández-Chacón
2The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390
3Department of Medical Physiology and Biophysics, School of Medicine, University of Seville, 41009 Seville, Spain
Thomas C. Südhof
2The Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, UT Southwestern Medical Center, Dallas, TX 75390
Erwin Neher
1Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany
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: [email protected]
Abbreviations used in this paper: NP-EGTA, nitrophenyl-EGTA; RRP, readily releasable pool; SNARE, soluble N-ethylmaleimide–sensitive fusion protein receptor; SRP, slowly releasable pool; WT, wild-type.
Received:
April 24 2003
Accepted:
July 17 2003
Online ISSN: 1540-7748
Print ISSN: 0022-1295
The Rockefeller University Press
2003
J Gen Physiol (2003) 122 (3): 265–276.
Article history
Received:
April 24 2003
Accepted:
July 17 2003
Citation
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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