SNARE Protein Dynamics during Synaptic Transmission

突触传递过程中的 SNARE 蛋白质动力学

• 批准号: 7342772
• 负责人: ROBERT STEPHEN ZUCKER
• 金额: $ 32.93万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7342772
• 关键词: Action Potentials; Arsenicals; Binding; C-terminal; Calcium; Calcium Channel; Cell membrane; Cells; Chemicals; Chromosome Pairing; Closure; Complex; Cyan Fluorescent Protein; Cytoplasm; Detection; Development; Dissociation; Docking; Endocytosis; Exocytosis; Fluorescence; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Foundations; Functional disorder; Goals; Hippocampus (Brain); Image; Individual; Label; Lateral; Life; Measurement; Measures; Melanocytic nevus; Membrane; Membrane Proteins; Microscopy; Mission; Modeling; Mole the mammal; Molecular; Molecular Machines; Molecular Motors; Monitor; Movement; N-terminal; Neighborhoods; Neurons; Neurotransmitters; Noise; Pathway interactions; Phospholipids; Procedures; Process; Protein Dynamics; Proteins; Protocols documentation; Rattus; Recovery; Recycling; Research Personnel; Running; SNAP receptor; Signal Transduction; Speed; Stimulus; Strategic Planning; Surface; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Testing; Therapeutic Intervention; Time; United States National Institutes of Health; VAMP-2; Vesicle; base; improved; innovation; nervous system disorder; postsynaptic; presynaptic; programs; receptor; soluble NSF attachment protein; spatial relationship; synaptic function; synaptotagmin; syntaxin; syntaxin 1; tool; two-photon; vesicle-associated membrane protein

DESCRIPTION (provided by applicant): The synapse is the point of functional contact between neurons, where information is communicated between cells. Each electrical impulse in the presynaptic neuron leads to the secretion of chemical neurotransmitter, which is packaged at the presynaptic active zone in small membrane-bound vesicles. Fusion of the vesicles with the plasma membrane results in liberation of transmitter molecules, which bind postsynaptically to receptor channels and signal the postsynaptic cell of the presence of presynaptic activity. Vesicle fusion is accomplished by a molecular machine consisting of several components. Vesicles are docked at the membrane by the SNARE complex of vesicular membrane protein VAMP (vesicle associated membrane protein) and the plasma membrane proteins syntaxin and SNAP-25 (soluble NSF-attachment protein of 25 kD). Presynaptic impulses open calcium channels to admit calcium to the cytoplasm, where it binds to the vesicular protein synaptotagmin, which interacts with SNAREs and the plasma membrane to initiate fusion. SNAREs must assemble before arrival of action potentials in order for vesicles to be primed for release. After fusion, SNAREs must disassemble before the vesicle membrane is recovered by endocytosis and recycled for reuse. Some models of vesicle fusion involve the tightening of the SNARE complex as an early step in fusion. The timing of these processes is unknown, and they have never been measured directly. This project will use fluorescence resonance energy transfer (FRET) interactions between fluorescent tags on vesicle and plasma membrane components of SNAREs to characterize their assembly, disassembly, and conformational changes on fusion. Specifically, cerulean-SNAP-25B and citrine-VAMP-2 N-terminal interactions, and VAMP-2-cerulean and syntaxin-1-citrine C-terminal interactions, will be studied in cultured rat hippocampal neurons under field stimulation. FRET will be measured by enhanced exciter emission or by reduced donor emission on donor excitation, and by two-photon fluorescence lifetime imaging microscopy (FLIM). The dispersion and re-aggregation of SNAP-25 following secretion will also be analyzed. This project will advance the specific goal of the NIH Roadmap of developing innovative tools to study interactions between individual proteins within single cells. Understanding the molecular mechanisms of synaptic function provides an essential foundation for the development of rational and effective therapeutic interventions to treat synaptic dysfunctions underlying numerous neurological disorders. Understanding the molecular machinery of synaptic transmission is a specific priority of the NINDS Strategic Plan in furtherance of the NINDS primary mission of reducing the burden of neurological disease.

描述（由申请人提供）：突触是神经元之间的功能接触点，在此细胞之间进行信息交流。突触前神经元中的每个电脉冲导致化学神经递质的分泌，其被包装在突触前活动区的小膜结合囊泡中。囊泡与质膜的融合导致递质分子的释放，递质分子在突触后与受体通道结合，并向突触后细胞发出突触前活动存在的信号。囊泡融合是通过由多个组件组成的分子机器完成的。囊泡通过囊泡膜蛋白VAMP（囊泡相关膜蛋白）和质膜蛋白突触融合蛋白和SNAP-25（25 kD的可溶性NSF附着蛋白）的SNARE复合物停靠在膜上。突触前冲动打开钙通道，使钙进入细胞质，在那里它与囊泡蛋白突触结合蛋白结合，后者与SNARE和质膜相互作用，启动融合。SNARE必须在动作电位到达之前组装，以便囊泡被引发释放。融合后，SNARE必须分解，然后囊泡膜才能通过内吞作用恢复并回收再利用。囊泡融合的一些模型涉及收紧SNARE复合物作为融合的早期步骤。这些过程的时间是未知的，他们从来没有被直接测量。本项目将利用荧光共振能量转移（FRET）的囊泡和质膜组件的SNARE的荧光标记之间的相互作用，以表征它们的组装，拆卸，融合和构象变化。具体而言，将在场刺激下在培养的大鼠海马神经元中研究天蓝色-SNAP-25 B和柠檬酸-VAMP-2 N-末端相互作用，以及VAMP-2-天蓝色和突触融合蛋白-1-柠檬酸C-末端相互作用。FRET将通过增强的激发发射或通过供体激发时减少的供体发射以及通过双光子荧光寿命成像显微镜（FLIM）来测量。还将分析分泌后SNAP-25的分散和再聚集。该项目将推进NIH路线图的具体目标，即开发创新工具来研究单细胞内单个蛋白质之间的相互作用。了解突触功能的分子机制为开发合理有效的治疗干预措施以治疗众多神经系统疾病的突触功能障碍提供了重要基础。了解突触传递的分子机制是NINDS战略计划的一个具体优先事项，以促进NINDS减轻神经系统疾病负担的主要使命。