The Molecular Mechanism Of Munc18-1 And Munc13-1 In Neuronal SNARE Complex Formation And Synaptic Exocytosis

The brain is the origins of emotions,memories,and thoughts.The neuron is the basic unit for these processes.Neurons have a specialized structure called axon,which could carry and transmit electric signals from the soma to the nerve terminal.Electric signals activate chemical signals to release from the nerve terminal to downstream organs or effectors through a specialized structure called the synapse.The synapse is a structure made up of axon terminal diverticulum(pre-synapse),dendrites or somas of the next neuron or effector(post-synapse),and an intercellular substance gap between the two structures called synaptic cleft.Pre-synaptic terminal contains masses of synaptic vesicles,which is loaded by several of neurotransmitters.Once the electric signals reach axon terminal,the voltage-gated calcium channels are activated and extracellular calcium will flow into pre-synaptic terminal.In response to the calcium signal,synaptic vesicles will fuse with pre-synaptic membrane and release the neurotransmitters into synaptic cleft.The neurotransmitters diffuse in synaptic cleft and finally bind to specific receptors on post-synaptic membrane;therefore,the signals are transduced to the next neuron or effector.The synaptic vesicles,which are loaded with neurotransmitters,undergo tethering,docking,priming,and fusion steps to release the neurotransmitters into synaptic cleft.The neuronal soluble N-ethylmaleimide sensitive factor attachment protein receptors(SNAREs)Syntaxin-1,SNAP-25 and Synaptobrevin-2/VAMP2 are believed to be the basic machinery for synaptic exocytosis.The three SNARE proteins could assemble into a four helical bundle complex called the SNARE complex,which brings synaptic vesicle and pre-synaptic membrane together and mediates membrane fusion.For nervous system,exquisite regulations are necessary.Perhaps the most important regulators are Sec1/Munc18 family protein Munc18-1 and Unc13/Munc13 s family protein Munc13-1.Although Munc18-1 and Munc13-1 null both cause severe neuron function and neurotransmitter release defects,the underlining mechanism remains unclear.For Munc13-1,functional relevance with SNARE proteins and Munc18-1 are enigmatic.Especially for the activation of Munc18-1/Syntaxin-1 complex and the initiation of SNARE complex assembly.In this thesis,we mainly focus on the fundamental function of Munc18-1 and Munc13-1 in mediating SNARE complex assembly and synaptic exocytosis,the conclusions are as follow:i)Determined the crystal structure of Munc13-1 MUN domain and distinguished four subdomains of the MUN domain;found a hydrophobic core located at the junction of subdomain B and C,which is the active center of Munc13-1 MUN domain;ii)Expounded the molecular mechanism of Munc13-1 MUN domain-mediated Syntaxin-1 opening;discovered the direct interacting site of Munc13-1 MUN domain on Syntaxin-1;iii)Determined the crystal structure of Munc13-1 MUN domain and Synaptobrevin-2/VAMP2 complex;proposed a synaptic vesicle priming complex,which contains Munc18-1/Syntaxin-1 complex,Synaptobrevin-2/VAMP2,and Munc13-1 MUN domain;In conclusion,this thesis systematically analyzed and revealed the molecular mechanism of Munc13-1 and Munc18-1 in neuronal SNARE complex formation,synaptic vesicle priming,and synaptic exocytosis.Our results established the foundations and shed new light on the understanding of the molecular mechanisms of learning,thinking,and memory of the brain.