Dissecting the Synergistic Roles of Synaptotagmin and Complexin in Ca2+-Regulated Exocytosis

Abstract

Controlled release of neurotransmitters stored in synaptic vesicles (SV) is central to all information processing in the brain. This process relies on the efficient coupling of SV fusion to the triggering signal - action potential evoked Ca2+ influx. SV fusion is catalysed by the synaptic SNARE proteins: VAMP2 on the SV (v-SNAREs) and syntaxin and SNAP25 on the pre-synaptic membrane (t-SNAREs). The fusion is tightly regulated by Complexin (Cpx) and presynaptic Ca2+ release sensor Synaptotagmin1 (Syt1) to enable ultra-fast Ca2+-coupled release. However, the precise molecular mechanisms of this process remain enigmatic. To address this, we used the recently developed pore-spanning lipid bilayer system, which allows for the precision study of the sub-steps of individual vesicle fusion events at tens of milliseconds timescale. We find that Syt1 and Cpx act co-operatively to generate and maintain a Ca2+-sensitive pool of vesicle under in vitro conditions. Our data suggest that oligomers of Syt1 engage a limited number of ‘central’ SNAREpins via the ‘primary’ binding site to introduce an un-stable clamp. This ‘delay’ in the fusion process enables Cpx to arrests the assembly of remainder ‘ectopic’ SNAREpins, in turn producing a stable clamped state. These docked vesicles are triggered to fuse upon Ca2+ influx and activation of Syt1-associated SNAREpins are sufficient to elicit a rapid (0.1s), synchronous fusion. Overall, our suggests that Syt1 plays a central role in orchestrating Ca2+-coupled neurotransmitter release.