A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release
Neurotransmiter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a primary interface comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper. Significance statementNeurotransmiter release requires SNARE complexes that fuse synaptic vesicles with the plasma membrane and the Ca2+-sensor synaptotagmin-1, which was thought to facilitate membrane fusion directly through its Ca2+-binding loops. However, binding of Synaptotagmin-1 to SNARE complexes orients these loops away from the fusion site. Using molecular dynamics simulations, we show that placing Synaptotagmin-1 at the fusion site hinders the action of SNARE complexes. Spectroscopic studies show that Ca2+ binding to Synaptotagmin-1 can change its interactions with SNARE complexes and, together with molecular modeling, suggest that Synaptotagmin-1 acts as a lever, pulling SNARE complexes and thus facilitating their action on the membranes to induce fusion. Functional studies described in the accompanying paper support this hypothesis.