Ca²⁺-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes

The Ca²⁺ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving 'polybasic', 'primary' and 'tripartite' interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca²⁺-free synaptotagmin-1 binds to SNARE complexes anchored on PIP₂-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca²⁺ induces tight binding of synaptotagmin-1 to PIP₂-containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca²⁺-dependent synaptotagmin-1-PIP₂-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca²⁺ releases this interaction, inducing PIP₂/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.