Synaptotagmins I and IV promote transmitter release independently of Ca2+ binding in the C2A domain

At nerve terminals, a focal and transient increase in intracellular Ca2+ triggers the fusion of neurotransmitter-filled vesicles with the plasma membrane. The most extensively studied candidate for the Ca2+-sensing trigger is synaptotagmin I, whose Ca2+-dependent interactions with acidic phospholipids and syntaxin(1) have largely been ascribed to its C(2)A domain(2-6), although the C2B domain also binds Ca2+ (refs 7, 8). Genetic tests of synaptotagmin I have been equivocal as to whether it is the Ca2+-sensing trigger of fusion(6,9-15). Synaptotagmin IV, a related isoform that does not bind Ca2+ in the C(2)A domain, might be an inhibitor of release(16,17). We mutated an essential aspartate of the Ca2+ binding site of the synaptotagmin I C(2)A domain and expressed it in Drosophila lacking synaptotagmin I. Here we show that, despite the disruption of the binding site, the Ca2+-dependent properties of transmission were not altered. Similarly, we found that synaptotagmin IV could substitute for synaptotagmin I. We conclude that the C(2)A domain of synaptotagmin is not required for Ca2+-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.