The role of the C(2)A domain of synaptotagmin 1 in asynchronous neurotransmitter release

Following nerve stimulation, there are two distinct phases of Ca(2+)-dependent neurotransmitter release: a fast, synchronous release phase, and a prolonged, asynchronous release phase. Each of these phases is tightly regulated and mediated by distinct mechanisms. Synaptotagmin 1 is the major Ca(2+) sensor that triggers fast, synchronous neurotransmitter release upon Ca(2+) binding by its C(2)A and C(2)B domains. It has also been implicated in the inhibition of asynchronous neurotransmitter release, as blocking Ca(2+) binding by the C(2)A domain of synaptotagmin 1 results in increased asynchronous release. However, the mutation used to block Ca(2+) binding in the previous experiments (aspartate to asparagine mutations, syt(D-N)) had the unintended side effect of mimicking Ca(2+) binding, raising the possibility that the increase in asynchronous release was directly caused by ostensibly constitutive Ca(2+) binding. Thus, rather than modulating an asynchronous sensor, syt(D-N) may be mimicking one. To directly test the C(2)A inhibition hypothesis, we utilized an alternate C(2)A mutation that we designed to block Ca(2+) binding without mimicking it (an aspartate to glutamate mutation, syt(D-E)). Analysis of both the original syt(D-N) mutation and our alternate syt(D-E) mutation at the Drosophila neuromuscular junction showed differential effects on asynchronous release, as well as on synchronous release and the frequency of spontaneous release. Importantly, we found that asynchronous release is not increased in the syt(D-E) mutant. Thus, our work provides new mechanistic insight into synaptotagmin 1 function during Ca(2+)-evoked synaptic transmission and demonstrates that Ca(2+) binding by the C(2)A domain of synaptotagmin 1 does not inhibit asynchronous neurotransmitter release in vivo.