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

Abstract

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 syntaxin¹ have largely been ascribed to its C₂A domain^2,3,4,5,6, 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^{6,9,10,11,12,13,14,15}. Synaptotagmin IV, a related isoform that does not bind Ca²⁺ in the C₂A domain, might be an inhibitor of release^16,17. We mutated an essential aspartate of the Ca²⁺-binding site of the synaptotagmin I C₂A 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 C₂A domain of synaptotagmin is not required for Ca²⁺-dependent synaptic transmission, and that synaptotagmin IV promotes rather than inhibits transmission.