Ca2+-dependent, phospholipid-binding residues of synaptotagmin are critical for excitation-secretion coupling in vivo.

Synaptotagmin I is the Ca2+ sensor for fast, synchronous release of neurotransmitter; however, the molecular interactions that couple Ca2+ binding to membrane fusion remain unclear. The structure of synaptotagmin is dominated by two C-2 domains that interact with negatively charged membranes after binding Ca2+. In vitro work has implicated a conserved basic residue at the tip of loop 3 of the Ca2+-binding pocket in both C-2 domains in coordinating this electrostatic interaction with anionic membranes. Although results from cultured cells suggest that the basic residue of the C(2)A domain is functionally significant, such studies provide contradictory results regarding the importance of the C2B basic residue during vesicle fusion. To directly test the functional significance of each of these residues at an intact synapse in vivo, we neutralized either the C(2)A or the C2B basic residue and assessed synaptic transmission at the Drosophila neuromuscular junction. The conserved basic residues at the tip of the Ca2+-binding pocket of both the C(2)A and C2B domains mediate Ca2+-dependent interactions with anionic membranes and are required for efficient evoked transmitter release. Our results directly support the hypothesis that the interactions between synaptotagmin and the presynaptic membrane, which are mediated by the basic residues at the tip of both the C(2)A and C2B Ca2+-binding pockets, are critical for coupling Ca2+ influx with vesicle fusion during synaptic transmission in vivo. Our model for synaptotagmin's direct role in coupling Ca2+ binding to vesicle fusion incorporates this finding with results from multiple in vitro and in vivo studies.