Fast resupply of synaptic vesicles requires synaptotagmin-3.

Sustained neuronal activity demands a rapid resupply of synaptic vesicles to maintain reliable synaptic transmission. Such vesicle replenishment is accelerated by submicromolar presynaptic Ca<sup>2+</sup> signals by an as-yet unidentified high-affinity Ca<sup>2+</sup> sensor<sup>1,2</sup>. Here we identify synaptotagmin-3 (SYT3)<sup>3,4</sup> as that presynaptic high-affinity Ca<sup>2+</sup> sensor, which drives vesicle replenishment and short-term synaptic plasticity. Synapses in Syt3 knockout mice exhibited enhanced short-term depression, and recovery from depression was slower and insensitive to presynaptic residual Ca<sup>2+</sup>. During sustained neuronal firing, SYT3 accelerated vesicle replenishment and increased the size of the readily releasable pool. SYT3 also mediated short-term facilitation under conditions of low release probability and promoted synaptic enhancement together with another high-affinity synaptotagmin, SYT7 (ref. <sup>5</sup>). Biophysical modelling predicted that SYT3 mediates both replenishment and facilitation by promoting the transition of loosely docked vesicles to tightly docked, primed states. Our results reveal a crucial role for presynaptic SYT3 in the maintenance of reliable high-frequency synaptic transmission. Moreover, multiple forms of short-term plasticity may converge on a mechanism of reversible, Ca<sup>2+</sup>-dependent vesicle docking.