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 C₂ domains and the Ca²⁺-independent bottom face of the C₂B domain. However, the functional importance of the C ₂B 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 C ₂B 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.