Unraveling the Functions of Synaptotagmin and Munc13 in Neurotransmitter Release

Neurotransmitter release is a central event in interneuronal communication. The release machinery includes three SNAREs (soluble N-ethylmaleimide sensitive factor adaptor protein receptor) and Munc18-1 as core components. Munc13, synaptotagmin and complexin underlie the exquisitely tight regulation of synaptic exocytosis. The SNAREs form the SNARE complex that brings synaptic vesicles and plasma membrane together. This complex is disassembled by NSF/α-SNAP (soluble N-ethylmaleimide sensitive fusion protein/NSF attachment protein). Munc18 binds to syntaxin, which keeps syntaxin in its closed confirmation and prevents to form the SNARE complex. Interactions between Munc13, Munc18, and syntaxin perform a vital role in regulation of the SNARE complex formation. Tight regulation of the release machinery requires other factors such as Ca2+ sensor Synaptotagmin-1 and negatively charged lipids. Synaptotagmin interacts with the SNARE complex and the negatively charged lipids, and initiates the synchronous fast release by sensing the Ca2+ influx. It is crucial to investigate functions of individual proteins to understand the mechanism of membrane fusion and neurotransmitter release. Therefore, we investigated the mechanism of membrane bridging by synaptotagmin. Our cryo-electron microscopy (cryo-EM) images showed that a majority of synaptotagmin fragment containing both C¬2A and C2B domains (C2AB) molecules bridge membranes directly. Fluorescence spectroscopy demonstrates that the bottom of the C2B domain contacts the membrane in a substantial population of membrane-bound synaptotagmin fragments. NMR analysis of C2AB-nanodiscs shows that a fraction of C2AB molecules binds to membranes with antiparallel orientations of the C2 domains. Together with previous studies, these results show that direct bridging constitutes the prevalent mechanism of membrane bridging by synaptotagmin, suggesting that this mechanism underlies the function of synaptotagmin-1 in neurotransmitter release. We have also discovered that Munc13-1 can bridge membranes in a Ca2+-independent manner, which shed light to the docking activity of Munc13-1. We also showed that Munc13-1 can cause efficient lipid mixing and slow content mixing together with Munc18-1 in the absence of synaptotagmin-1. Addition of synaptotagmin facilitates the fusion pore formation in content mixing assays. Recently, we were able to reconstitute key components of the release machinery. We are further investigating our observations with cryo-electron microscopy to understand the mechanism of membrane fusion.