Structural Basis of Quantal Release

量子释放的结构基础

• 批准号: 9900872
• 负责人: Fei Li
• 金额: $ 12.12万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9900872
• 关键词: Address; Affinity; Allosteric Regulation; Award; Biological; Brain; Brain Diseases; Carrier Proteins; Cells; Chemicals; Communication; Communities; Complex; Coupling; Cryo-electron tomography; Cryoelectron Microscopy; Disease; Electron Microscopy; Electrophysiology (science); Endocytosis; Environment; Equilibrium; Event; Exhibits; Exocytosis; Foundations; Future; Glutamate Transporter; Glutamates; Goals; Institution; Ions; Knowledge; Learning; Lysosomes; Membrane Potentials; Membrane Proteins; Membrane Transport Proteins; Mentors; Methodology; Mitochondria; Molecular; Neurologic; Neurons; Neurosciences; Neurotransmitters; Organelles; Pathway interactions; Physiological; Physiology; Play; Process; Property; Proteins; Proton Pump; Proton-Translocating ATPases; Rattus; Receptor Activation; Regulation; Research; Research Personnel; Research Project Grants; Research Training; Resolution; Resources; Role; Scientist; Signal Transduction; Structure; Structure-Activity Relationship; Synapses; Synaptic Transmission; Synaptic Vesicles; Techniques; Technology; Testing; Therapeutic Intervention; Time; Training; Training Programs; Vesicle; Work; X-Ray Crystallography; Xenopus oocyte; brain health; career; driving force; insight; interest; mutant; neurotransmission; neurotransmitter transport; particle; protein complex; protein purification; protein structure function; quantum; receptor; skills; synaptic function; tool; training opportunity; uptake; vacuolar H+-ATPase

PROJECT SUMMARY/ABSTRACT Vesicular glutamate transporters (VGLUTs) package the major excitatory neurotransmitter glutamate into synaptic vesicles, thus play a critical role in quantal release and neurotransmission. Despite decades of research, the mechanism of glutamate transport by the VGLUTs and its regulation under different ionic and pH conditions during exocytosis and endocytosis remains poorly understood. The lack of molecular level understanding of the structure and function of VGLUTs severely hinders our ability to understand their role in normal brain function, as well as in many psychiatric and neurological conditions where malfunction of VGLUTs has been implicated. This proposal aims to bridge this gap by providing a molecular blue print of a mammalian VGLUT protein with atomic details. Specifically, the candidate Dr. Fei Li will determine high-resolution structures of the rat VGLUT1 (rVGLUT1) by cryo electronmicrosocpy (cryo-EM) (Aim 1, mentored) and characterize its function by electrophysiology (Aim 2, mentored). Dr. Fei Li will further determine the structure of the native synaptic vesicle by cryo electrontomography (cryo-ET) (Aim 3A, mentored) and to reveal the higher order organization and interactions of synaptic vesicle membrane proteins (Aim 3B, independent). The goal of this K99/R00 Pathway to Independence Award proposal is to enhance Dr. Li’s knowledge in neuroscience and to provide opportunity of additional training in several key techniques that are critical to launching her independent research career focusing on the structure and function of neuronal membrane proteins in a leading research institution. The proposed aims allow Dr. Li to explore new biological questions that she can continue to investigate in her independent career while providing opportunities for training at the same time. Dr. Li has a strong interest in membrane protein structure and function in the context of brain physiology, and a strong background in membrane protein and X-ray crystallography. To guide the proposed project and to enhance her career prospects, Dr. Li has selected three leading scientists in, respectively, membrane proteins (Dr. Robert Stroud), electron microscopy (Dr. Yifan Cheng), as well as electrophysiology and neuroscience (Dr. Robert Edwards) as her co-mentors. Training through this project will allow Dr. Li to build a highly versatile and integrative skill set extending her background in membrane proteins and X-ray crystallography to the state-of-the-art cryo-EM and cryo-ET technology. Skills in electrophysiology will provide her with the tools required for functional analysis. Together, the research project and training program proposed in this award will play a critical role in preparing Dr. Li for a successful independent research career.

项目总结/摘要 囊泡谷氨酸转运蛋白（VGLUT）将主要的兴奋性神经递质谷氨酸包装成 突触囊泡，因此在量子释放和神经传递中起关键作用。尽管经过数十年的 研究，VGLUTs转运谷氨酸的机制及其在不同离子和pH下的调节 胞吐和胞吞过程中的条件仍然知之甚少。缺乏分子水平 对VGLUT结构和功能的理解严重阻碍了我们理解它们在 正常的脑功能，以及许多精神和神经疾病，其中VGLUT功能障碍， 被牵连了这项提案旨在通过提供哺乳动物的分子蓝图来弥合这一差距。 具有原子细节的VGLUT蛋白质。具体来说，候选人李飞博士将确定高分辨率 大鼠VGLUT 1（rVGLUT 1）的冷冻电镜（cryo-EM）结构（目标1，指导）， 通过电生理学表征其功能（目标2，指导）。李飞博士将进一步确定 通过冷冻电子断层扫描（cryo-ET）（Aim 3A，指导）观察天然突触囊泡的结构， 揭示突触囊泡膜蛋白的高级结构和相互作用（目的 3B，独立）。这个K99/R 00独立之路奖提案的目标是提高李博士的 神经科学知识，并提供几个关键技术的额外培训机会， 这对她开始独立的研究生涯至关重要，她的研究重点是神经元的结构和功能。 膜蛋白在一个领先的研究机构。拟议的目标使李博士能够探索新的生物学 她可以在独立的职业生涯中继续调查的问题，同时提供机会， 训练同时。李博士对膜蛋白的结构和功能有着浓厚的兴趣 大脑生理学，以及膜蛋白和X射线晶体学的强大背景。引导 李博士在提出的项目和提高她的职业前景，选择了三个领先的科学家， 分别是膜蛋白（Robert Stroud博士）、电子显微镜（Yifan Cheng博士），以及 电生理学和神经科学（罗伯特·爱德华兹博士）作为她的共同导师。通过该项目进行的培训将 使李博士能够建立一个高度通用和综合的技能集，扩展她在膜蛋白方面的背景 和X射线晶体学到最先进的冷冻EM和冷冻ET技术。电生理学技能 将为她提供功能分析所需的工具。研究项目和培训 在这个奖项提出的计划将发挥关键作用，准备李博士成功的独立 研究生涯。