Concurrent Binding of Complexin and Synaptotagmin to Liposome-Embedded SNARE Complexes

[Image: see text] Synaptotagmin and complexin regulate SNARE-mediated synaptic vesicle exocytosis. It has been proposed that complexin clamps membrane fusion and that Ca(2+)-synaptotagmin displaces complexin from SNARE complexes to relieve this clamping activity. Using a reconstituted system, we demonstrate that complexin and synaptotagmin simultaneously bind to neuronal SNARE complexes and that both apo-synaptotagmin and complexin inhibit SNARE-mediated membrane fusion. Moreover, the clamping ability of apo-synaptotagmin occluded the clamping activity of complexin until the arrival of a Ca(2+) trigger, at which point synaptotagmin accelerated fusion while high concentrations of complexin inhibited fusion. Thus, the inhibitory patterns of synaptotagmin and complexin are different, suggesting that SNAREs assemble into distinct states along the fusion pathway. These data also suggest that during synaptotagmin-regulated vesicle−vesicle fusion, complexin does not function as a fusion clamp that is relieved by Ca(2+)-synaptotagmin.