Calcium Binding by Synaptotagmin's C₂A Domain is an Essential Element of the Electrostatic Switch That Triggers Synchronous Synaptic Transmission

Synaptotagmin is the major calcium sensor for fast synaptic transmission that requires the synchronous fusion of synaptic vesicles. Synaptotagmin contains two calcium-binding domains: C 2 A and C 2 B. Mutation of a positively charged residue (R233Q in rat) showed that Ca 2+ -dependent interactions between the C 2 A domain and membranes play a role in the electrostatic switch that initiates fusion. Surprisingly, aspartate-to-asparagine mutations in C 2 A that inhibit Ca 2+ binding support efficient synaptic transmission, suggesting that Ca 2+ binding by C 2 A is not required for triggering synchronous fusion. Based on a structural analysis, we generated a novel mutation of a single Ca 2+ -binding residue in C 2 A (D229E in Drosophila ) that inhibited Ca 2+ binding but maintained the negative charge of the pocket. This C 2 A aspartate-to-glutamate mutation resulted in ∼80% decrease in synchronous transmitter release and a decrease in the apparent Ca 2+ affinity of release. Previous aspartate-to-asparagine mutations in C 2 A partially mimicked Ca 2+ binding by decreasing the negative charge of the pocket. We now show that the major function of Ca 2+ binding to C 2 A is to neutralize the negative charge of the pocket, thereby unleashing the fusion-stimulating activity of synaptotagmin. Our results demonstrate that Ca 2+ binding by C 2 A is a critical component of the electrostatic switch that triggers synchronous fusion. Thus, Ca 2+ binding by C 2 B is necessary and sufficient to regulate the precise timing required for coupling vesicle fusion to Ca 2+ influx, but Ca 2+ binding by both C 2 domains is required to flip the electrostatic switch that triggers efficient synchronous synaptic transmission.