The role of the C2A 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 an artifact of ostensibly constitutive Ca 2+ binding. To directly test this 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 . Significance statement This study provides mechanistic insights into synaptotagmin function during asynchronous neurotransmitter release and supports a dramatically different hypothesis regarding the mechanisms triggering asynchronous vesicle fusion. Using two distinct C 2 A mutations that block Ca 2+ binding, we report opposing effects on synchronous, spontaneous, and asynchronous neurotransmitter release. Importantly, our data demonstrate that Ca 2+ binding by the C 2 A domain of synaptotagmin does not regulate asynchronous release and thus disprove the current inhibition hypothesis. We propose a spatial competition hypothesis to explain these seemingly discordant results of the differing C 2 A Ca 2+ binding mutations.