The Mechanisms Of Munc13 In Regulating SNAREs Mediated Vesicle Exocytosis

Neural signal was transducted by nervous system,which was one of the most complicated systems in human.Nervous system diseases,such as Alzheimer's disease,Autism and Epilepsy,caused by the abnormal neural activity severely affected human health and drove a series of social problems.Neurotransmitter release was mediated by calcium-dependent presynaptic vesicles and plasma membrane fusion.Therefore,understanding the regulation of vesicle release was one of most fundamental questions in neuroscience research.Signal had to be transferred rapidly in normal nerve system.SNARE complex mediated vesicle fusion,which was precisely regulated by many proteins,was the known major route for signal transduction.SNARE complex was considered to be the basic structure of synaptic secretion,which provided a driving force for the membrane fusion.Previous studies showed that,before the SNARE complex assembled,syntaxin firstly combined with Munc18.It was so called “closed” conformation and couldn't form SNARE complex.Munc13 was known the key protein that could change syntaxin from “closed” to “open” conformation,but the precise process was largely unclear.Here we tried to figure out the mechanisms of Munc13 in regulating SNAREs mediated vesicle exocytosis by investigating the interaction between Munc13 and other proteins.Munc13-1,as known as an important regulator for fast vesicle fusion,severely impaired neurotransmitter release with its deficiency.To discover the underlying mechanisms of Munc13-1 in fast vesicle fusion,we used RNA interference technology to suppress the endogenous Munc13 expression and monitor the vesicle release by whole-cell patch-clamp.We identified that the NF residues in MUN domain of Munc13 were the key points for opening the Munc18-Syntaxin complex,playing an important role in MUN domain involved in vesicle release.To discover the target protein of the NF residues,we preliminary identified Syntaxin-1 as the functional target of Munc13-1 by using FRET experiment and Syntaxin structure analysis in vitro,and narrowed down the linker region of Syntaxin-1(the sequence between Habc domain and H3 domain)as responsible sequence.We screened the linker region of syntaxin-1 and identified Arg151 and Ile155 resides were the possible interacting sites.In mouse neuronal cells,we revealed that the RI residues of syntaxin were extremely important for the vesicle release,by using whole-cell patch clamp and RNA interference technology.These results suggested that RI residues were the key interacting sites with Munc13-1 for opening Munc18-Syntaxin complex.In this paper,we clarified the mechanisms of Munc13-1 in facilitating fast neurotransmitter release via interacting with syntaxin-1 for the first time,and proposed a new model for the regulation of neurotransmitter release.When the eight amino acids hydrophobic pocket of Munc13 attacked the RI residues in the linker region of syntaxin,the H3 domain was released from Munc18-syntaxin complex to form SNAREs with SNAP-25 and synaptobrevin,and trigger vesicle fusion.The mechanisms of signal transduction are gone deeper and more evidence and uncovered in the pathogenesis of neuronal diseases.