The Janus-Faced Nature of the C(2)B Domain Is Fundamental for Synaptotagmin-1 Function

Synaptotagmin-1 functions as a Ca(2+) sensor in neurotransmitter release, and was proposed to act on both the synaptic vesicle and plasma membranes through interactions involving the Ca(2+)-binding top loops of its C(2) domains and the Ca(2+)-independent bottom face of the C(2)B domain. However, the functional importance of the C(2)B domain bottom face is unclear. We now show that mutating two conserved arginines at the C(2)B domain bottom face practically abolishes synchronous release in hippocampal neurons. Reconstitution experiments reveal that Ca(2+)/synaptotagmin-1 can dramatically stimulate the rate of SNARE-dependent lipid mixing/fusion and that the two-arginine mutation strongly impairs this activity. These results demonstrate that synaptotagmin-1 function depends critically on the bottom face of the C(2)B domain and strongly support the notion that synaptotagmin-1 triggers membrane fusion and neurotransmitter release by bringing the vesicle and plasma membranes together, much like the SNAREs do but in a Ca(2+)-dependent manner.