SNARE Protein Dynamics during Synaptic Transmission

突触传递过程中的 SNARE 蛋白质动力学

• 批准号: 8040018
• 负责人: ROBERT STEPHEN ZUCKER
• 金额: $ 32.32万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8040018
• 关键词: Action Potentials; Arsenicals; Binding; C-terminal; Calcium; Calcium Channel; Cell membrane; Cells; Chemicals; Complex; Cyan Fluorescent Protein; Cytoplasm; Detection; Development; Dissociation; Docking; Endocytosis; Exocytosis; Fluorescein; Fluorescence; Fluorescence Resonance Energy Transfer; Foundations; Functional disorder; Goals; Hippocampus (Brain); Image; Individual; Label; Lateral; Life; Measurement; Measures; Membrane; Membrane Proteins; Microscopy; Mission; Modeling; Mole the mammal; Molecular; Molecular Machines; Molecular Motors; Monitor; Movement; N-terminal; National Institute of Neurological Disorders and Stroke; Neighborhoods; Neurons; Neurotransmitters; Noise; Pathway interactions; Phospholipids; Procedures; Process; Protein Dynamics; Proteins; Protocols documentation; Rattus; Recovery; Recycling; Research Personnel; Running; S-nitro-N-acetylpenicillamine; 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，是细胞之间信息交流的地方。突触前神经元的每一个电脉冲都导致化学神经递质的分泌，这些递质被包装在突触前活跃区的小膜结合囊泡中。囊泡与质膜的融合导致释放递质分子，这些递质分子结合突触后受体通道，并向突触后细胞发出突触前活动存在的信号。囊泡融合是由一个由若干组分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syntaxin和SNAP-25（可溶性nsf -附着蛋白25 kD）的SNARE复合物停靠在膜上。突触前脉冲打开钙通道，使钙进入细胞质，在细胞质中与囊泡蛋白synaptotagmin结合，后者与SNAREs和质膜相互作用，启动融合。陷阱必须在动作电位到达之前组装，以便囊泡准备释放。融合后，SNAREs必须在囊泡膜通过内吞作用恢复并回收再利用之前分解。一些囊泡融合模型涉及SNARE复合体的收紧作为融合的早期步骤。这些过程的时间是未知的，它们从来没有被直接测量过。该项目将利用SNAREs囊泡和质膜组分上荧光标签之间的荧光共振能量转移（FRET）相互作用来表征它们的组装、拆卸和融合时的构象变化。具体来说，将在电场刺激下研究培养的大鼠海马神经元中cerulean- snap25b和柠檬酸- vamp -2 n端相互作用，以及VAMP-2-cerulean和syntaxin-1-citrine c端相互作用。FRET将通过增强激发器发射或在供体激发下减少供体发射和双光子荧光寿命成像显微镜（FLIM）来测量。还将分析SNAP-25分泌后的分散和重新聚集情况。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来治疗许多神经系统疾病背后的突触功能障碍提供了重要的基础。了解突触传递的分子机制是NINDS战略计划的一个特定优先事项，以促进NINDS减轻神经系统疾病负担的主要任务。

项目成果

Dance of the SNAREs: assembly and rearrangements detected with FRET at neuronal synapses.

• DOI: 10.1523/jneurosci.2337-12.2013
• 发表时间: 2013-03-27
• 期刊: The Journal of neuroscience : the official journal of the Society for Neuroscience
• 作者: Degtyar V;Hafez IM;Bray C;Zucker RS
• 通讯作者: Zucker RS

A general model of synaptic transmission and short-term plasticity.

• DOI: 10.1016/j.neuron.2009.03.025
• 发表时间: 2009-05-28
• 期刊: NEURON
• 影响因子: 16.2
• 作者: Pan, Bin;Zucker, Robert S.
• 通讯作者: Zucker, Robert S.