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The Journal of Neuroscience, October 1, 2002, 22(19):8438-8446
Structure/Function Analysis of Ca2+ Binding to the C2A Domain of Synaptotagmin 1
Rafael Fernández-Chacón1, 4, Ok-Ho Shin1, Andreas Königstorfer3, Maria F. Matos1, Alexander C. Meyer4, Jesus Garcia2, Stefan H. Gerber1, Josep Rizo2, Thomas C. Südhof1, and Christian Rosenmund4 1 Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, and 2 Departments of Biochemistry and Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111, and 3 Max-Planck-Institut für experimentelle Medizin and 4 biophysikalische Chemie, 37070 Göttingen, Germany
Synaptotagmin 1, a Ca2+ sensor for fast synaptic vesicle exocytosis, contains two C2 domains that form Ca2+-dependent complexes with phospholipids. To examine the functional importance of Ca2+ binding to the C2A domain of synaptotagmin 1, we studied two C2A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca2+ binding and Ca2+-dependent phospholipid binding by the isolated C2A domain. Both mutations, however, did not alter the apparent Ca2+ affinity of the double C2 domain fragment, although both decreased the tightness of the Ca2+/phospholipid/double C2 domain complex. When introduced into the endogenous synaptotagmin 1 gene in mice, the D232N and D238N mutations had no apparent effect on morbidity and mortality and caused no detectable alteration in the Ca2+-dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hippocampal neurons from knock-in mice revealed that neither mutation induced major changes in synaptic transmission. The D232N mutation, however, caused increased synaptic depression during repetitive stimulation, whereas the D238N mutation did not exhibit this phenotype. Our data indicate that Ca2+ binding to the C2A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C2 domains cooperate and may be partially redundant in Ca2+-dependent phospholipid binding. Moreover, although the apparent Ca2+ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca2+/phospholipid complex is not. Our data also demonstrate that subtle changes in the biochemical properties of synaptotagmin 1 can result in significant alterations in synaptic responses.
Key words: synaptotagmin; neurotransmitter release; exocytosis; C2 domain; Ca2+-binding site; synaptic plasticity
Copyright © 2002 Society for Neuroscience 0270-6474/02/22198438-09$05.00/0
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