Ca²+-evoked transmitter release shows a high dynamic range over spontaneous release. We investigated the role of the Ca²+ sensor protein, Synaptotagmin2 (Syt2), in both spontaneous and Ca²+-evoked release under direct control of presynaptic [Ca²+](i), using an in vivo rescue approach at the calyx of Held. Re-expression of Syt2 rescued the highly Ca²+ cooperative release and suppressed the elevated spontaneous release seen in Syt2 KO synapses. This latter release clamping function was partially mediated by the poly-lysine motif of the C₂B domain. Using an aspartate mutation in the C₂B domain (D364N) in which Ca²+ triggering was abolished but release clamping remained intact, we show that Syt2 strongly suppresses the action of another, near-linear Ca²+ sensor that mediates release over a wide range of [Ca²+](i). Thus, Syt2 increases the dynamic range of synapses by driving release with a high Ca²+ cooperativity, as well as by suppressing a remaining, near-linear Ca²+ sensor.
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