The Janus-faced nature of the C2B domain is fundamental for synaptotagmin-1 function

Synaptotagmin-1 functions as a Ca2+ sensor in neurotransmitter release and was proposed to act on both the synaptic vesicle and plasma membranes through interactions involving the Ca2+ binding top loops of its C-2 domains and the Ca2+-independent bottom face of the C2B domain. However, the functional importance of the C2B domain bottom face is unclear. We now show that mutating two conserved arginine residues at the C2B domain bottom face practically abolishes synchronous release in hippocampal neurons. Reconstitution experiments reveal that Ca2+-synaptotagmin-1 can dramatically stimulate the rate of SNARE-dependent lipid mixing, and that the two-arginine mutation strongly impairs this activity. These results demonstrate that synaptotagmin-1 function depends crucially on the bottom face of the C2B 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 Ca2+- dependent manner.