The C(2)A domain of synaptotagmin is an essential component of the calcium sensor for synaptic transmission

The synaptic vesicle protein, synaptotagmin, is the principle Ca(2+) sensor for synaptic transmission. Ca(2+) influx into active nerve terminals is translated into neurotransmitter release by Ca(2+) binding to synaptotagmin’s tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca(2+)-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C(2)B) is critical for fusion, while the other domain (C(2)A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C(2)A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C(2)A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C(2)A mutation to date, demonstrating that the C(2)A domain is absolutely essential for synaptotagmin’s function as the electrostatic switch.