Neurotransmitter release in synapses underlies major brain functions such as cognition, emotion, and memory. The precise temporal control of the release is essential for healthy brain activities. SNAREs are known to be the core fusion machinery in neuro-exocytotic pathways. SNARE complex formation in the chasm of two membranes is mediated by cognate coiled-coil motifs on v- and t-SNAREs: One such motif from t-SNARE syntaxin 1A, two from t-SNARE SNAP-25, and another from v-SNARE VAMP2 all form a parallel four-stranded coiled coil that brings about the apposition of two membranes. However, The SNARE proteins do not have the required regulatory function to confer temporal on/off switching capability that requires other regulatory proteins like Munc13/Munc18, complexin, synaptotagmin, and so on. Moreover, a relationship has been discovered between the malfunction of neurotransmitter-release machinery and neuro-degeneration diseases. However, the mechanisms of just how these regulators and neuro-degeneration related proteins mediate synaptic vesicle exocytosis are still unknown.

In our work, we developed site-specific fluorescence resonance energy transfer (FRET) to investigate the formation of trans SNAREpin between complimentary t- and v-SNAREs reconstituted to separate proteoliposomes. In combination with the traditional bulk lipid mixing assay, we first found that α-synulcein may inhibit SNARE-dependent vesicle docking through membrane binding without affecting trans SNARE assembly. Moreover, C2AB and Ca2+ drive SNARE zippering at the membrane proximal region, although C2AB is less sufficient to stimulation fusion pore opening than Syt1. Third, by studying full-length Syt1, we demonstrated that for Syt1 to function as a major Ca2+ sensor for neuro-exocytosis, vectorial surface-charge asymmetry is required, and this may play a role in steering Syt1 to achieve productive trans binding to the plasma membrane rather than non-productive cis binding to the vesicle. Fourth, to study the fusion-pore opening step of the fusion process, we developed large DNA-probe-based content mixing to report pore expansion. Together with small-probe sulforhodamine-based content mixing to indicate pore opening, we showed that SNARE alone can inefficiently open the fusion pore, while Syt1/Ca2+ is efficient in opening the fusion pore but with relatively slow pore expansion. Finally, our results suggest that, in the Syt1 linker region, charged amino acids are asymmetrically-located, providing the Syt1 linker region with both the flexibility for vesicle docking and rigidity for opening the fusion pore.