Synaptotagmin 1, a Ca 2ϩ sensor for fast synaptic vesicle exocytosis, contains two C 2 domains that form Ca 2ϩ -dependent complexes with phospholipids. To examine the functional importance of Ca 2ϩ binding to the C 2 A domain of synaptotagmin 1, we studied two C 2 A domain mutations, D232N and D238N, using recombinant proteins and knock-in mice. Both mutations severely decreased intrinsic Ca 2ϩ binding and Ca 2ϩdependent phospholipid binding by the isolated C 2 A domain. Both mutations,

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... did not alter the apparent Ca 2ϩ affinity of the double C 2 domain fragment, although both decreased the tightness of the Ca 2ϩ /phospholipid/double C 2 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 Ca 2ϩ -dependent properties of synaptotagmin 1. Electrophysiological recordings of cultured hip-pocampal 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 Ca 2ϩ binding to the C 2 A domain of synaptotagmin 1 may be important but not essential, consistent with the finding that the two C 2 domains cooperate and may be partially redundant in Ca 2ϩdependent phospholipid binding. Moreover, although the apparent Ca 2ϩ affinity of the synaptotagmin 1/phospholipid complex is critical, the tightness of the Ca 2ϩ /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.