Structure/function analysis of Ca²⁺ binding to the C₂A domain of synaptotagmin 1

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.