Independent Yet Synergistic Roles of Synaptotagmin-1 and Complexin in Calcium Regulated Neuronal Exocytosis

Calcium (Ca2+)-evoked release of neurotransmitters from synaptic vesicles requires mechanisms both to prevent un-initiated fusion of vesicles (clamping) and to trigger fusion following Ca2+-influx. The principal components involved, namely the vesicular fusion machinery (SNARE proteins) and the regulatory proteins (Synaptotagmin-1 and Complexin) are well-known. Here, we use a reconstituted single-vesicle fusion assay to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act independently but synergistically to establish Ca2+-regulated fusion. Under physiologically-relevant conditions, we find that Synaptotagmin-1 oligomers bind and clamp a limited number of central SNARE complexes via the primary binding interface, to introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to independently arrest the remaining free peripheral SNAREpins to produce stably clamped vesicles. Activation of the central SNAREpins associated with Synaptotagmin-1 by Ca2+ is sufficient to trigger rapid (<100 msec) and synchronous fusion of the docked vesicles.