In neurons, SNAREs mediate membrane fusion between synaptic vesicles and plasma membranes to transfer neurotransmitters under regulation of other synaptic proteins in the synapse. It has previously been thought that there are distinct stages in membrane fusion events. First of all, synaptic vesicles are docked to the plasma membrane and trans-SNAREpin complexes are formed between two membranes while complete membrane fusion such as pore opening is prevented until the arrival of sufficient regulations. At the proper time, SNARE complexes form fully-extended cis-conformations, then consequently mix membranes into a continuous bilayer and open a pore for the neurotransmitter release.

In a normal synapse, the fusion event mediated by SNAREs must be regulated in timely fashion. Synaptotagmin-1 (syt1) is a Ca2+ sensor in the synaptic vesicle characterized by an N-terminal transmembrane domain, a flexible liner, and tandem C2 domains, C2AB, at the C-terminus. Syt1 and Ca2+ influx are well-known regulators for the docking of synaptic vesicles to the plasma membrane and also consequent fusion pore opening, although the specific mechanism is still unclear. SNARE-controlled membrane fusion events also depend on Sec1/Munc18 (SM) proteins. Munc18-1 is known to strongly bind to the closed syntaxin-1a conformation and Munc13 may play a role in the structural transition of syntaxin-1a to facilitate the formation of the t-SNARE complex. Complexin1 (cpx1) is a small protein containing four distinct functional regions. It binds to SNARE complexes and assists syt1 by simultaneously activating and clamping the core fusion machinery.

In the current work described here, we have established a new platform of trans-SNAREpin study tool using nanodiscs. SNARE-incorporated nanodiscs are able to trap trans-conformation of SNAREpin without further full fusion processes. Single-molecule fluorescence resonance energy transfer (smFRET) based on the total internal reflection microscopy (TIR) and electron paramagnetic resonance (EPR) were recruited to investigate structural information about trans-SNAREpin trapped in nanodiscs and SNARE-mediated fusogenic efficiencies in the presence of synaptic regulatory proteins. We first confirmed the presence of multiple conformations of trans-SNAREpin complex based on the new platform. We were then able to observe the role of each synaptic regulatory protein, such as Munc18-1 and cpx1, in the presence of syt1 during SNARE complex assembly and membrane fusion processes. Our results suggest that syt1 plays an overall role of regulation in SNARE-mediated synaptic vesicle fusion, including docking, membrane mixing, and fusion pore opening, while the other regulators Munc18-1 and cpx1 may also serve specific functions during this process. Munc18-1 was able to accelerate the complete SNAREpin formation and SNARE-mediated lipid mixing only in the absence of syt1, while cpx1 maintained the same function independent of syt1. Furthermore, content-mixing assays confirmed cpx1 N-terminal region acted as an active assistant for syt1 in Ca2+ triggered fusion pore-opening while Munc18-1 did not affect the result.

Considering the evolutionary-conserved function of SM proteins as activators in intracellular membrane fusion controlled by SNAREs and the experimental results in this work, it appears that lately-evolved membrane fusion systems such as synaptic exocytosis might fire Munc18-1 and recruit new controllers, syt1 and cpx1, to perform temporal and spatial regulation of sophisticated signal transmission.