Abstract
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 in these processes are the vesicular fusion machinery (SNARE proteins) and the regulatory proteins, Synaptotagmin-1 and Complexin. Here, we use a reconstituted single-vesicle fusion assay under physiologically-relevant conditions to delineate a novel mechanism by which Synaptotagmin-1 and Complexin act synergistically to establish Ca2+-regulated fusion. We find that under each vesicle, Synaptotagmin-1 oligomers bind and clamp a limited number of 'central' SNARE complexes via the primary interface and introduce a kinetic delay in vesicle fusion mediated by the excess of free SNAREpins. This in turn enables Complexin to arrest the remaining free 'peripheral' SNAREpins to produce a stably clamped vesicle. 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.
Highlights
The controlled yet rapid release of neurotransmitters stored in synaptic vesicles (SVs) is central to all information processing in the brain (Sudhof, 2013; Kaeser and Regehr, 2014; Rizo, 2018)
We examined if Syt1 and Cpx act on the same SNARE complexes sequentially or if they function separately to produce molecularly-distinct clamped SNAREpins under the same docked vesicles
We report that Syt1 and Cpx act concomitantly to clamp SNARE-driven constitutive fusion events (Figure 1)
Summary
The controlled yet rapid (sub-millisecond) release of neurotransmitters stored in synaptic vesicles (SVs) is central to all information processing in the brain (Sudhof, 2013; Kaeser and Regehr, 2014; Rizo, 2018). SV fusion is catalyzed by synaptic SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, VAMP2 on the vesicles (vSNAREs) and Syntaxin/SNAP25 (t-SNAREs) on the pre-synaptic membrane (Sollner et al, 1993; Weber et al, 1998). The prevailing theory is that in a ‘releaseready’ vesicle, multiple SNARE complexes are firmly held (‘clamped’) in a partially assembled state These ‘SNAREpins’ are synchronously released by Ca2+ to drive fusion dramatically faster than any one SNARE alone (Rothman et al, 2017; Brunger et al, 2018; Volynski and Krishnakumar, 2018). Several lines of evidence suggest that two synaptic proteins, Synaptotagmin-1 (Syt1) and Complexin (Cpx) play a critical role in establishing Ca2+-regulated neurotransmitter release (Geppert et al, 1994; Xu et al, 2007; Bacaj et al, 2013; Yang et al, 2013; Huntwork and Littleton, 2007)
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