Structural Mechanism of the Acetylcholine Receptor in Tubular Membrane Crystals

管状膜晶体中乙酰胆碱受体的结构机制

• 批准号: 8102674
• 负责人: PETER Nigel UNWIN
• 金额: $ 23.69万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8102674
• 关键词: Acetylcholine; Affect; Alzheimer' s Disease; Anesthetics; Binding; Binding Sites; Biological; Brain; Chemical Synapse; Cholinergic Receptors; Collaborations; Communication; Cryoelectron Microscopy; Development; Disease; Electric Organ; Electron Microscopy; Electrons; Environment; Epilepsy; Event; Exposure to; Family; Family member; Film; Freezing; Funding; Future; Gated Ion Channel; Goals; Helium; Image; Ion Channel; Life; Lipids; Liquid substance; Mediating; Membrane; Membrane Proteins; Mental Depression; Mental disorders; Methodology; Methods; Microscopic; Modeling; Molecular; Myasthenia Gravis; Nature; Nervous system structure; Neurons; Neurotransmitters; Nicotine Dependence; Nicotinic Receptors; Optics; Performance; Peripheral Nervous System; Pharmaceutical Preparations; Pharmacologic Substance; Physiological; Process; Property; Proteins; Radiation; Reaction; Research; Research Institute; Resolution; Schizophrenia; Signal Transduction; Specimen; Staging; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; Torpedo; Tube; Tubular formation; Work; aqueous; computer program; human disease; improved; in vivo; insight; member; membrane model; neuromuscular; neuromuscular transmission; programs; receptor; receptor binding; receptor function; user-friendly

DESCRIPTION (provided by applicant): The nicotinic Acetylcholine (ACh) receptor mediates fast synaptic transmission by converting transiently to an open-channel form when activated by ACh released into the synaptic cleft. Our goal is to understand the structural transition underlying this event, i.e. to provide a definitive molecular picture of how the channel works. The ACh receptor-rich membranes for our studies come from the electric organ of the Torpedo ray, and we analyze tubular membrane crystals grown from them by cryo-electron microscopy. The proposed research builds on a long-term programmed of technical development, which recently yielded a refined atomic model of the membrane-bound receptor in the closed-channel form. To investigate the open-channel structure we will propel acetylcholine-containing spray droplets onto the tubular crystals lying within a thin aqueous film on the e.m. grid and then trap the reaction (within ~2ms) by plunge-freezing. By this means we recapitulate the brief reaction of neurotransmitter with receptors at the synapse and capture the opened channels in both a physiological ionic environment and a native membrane setting. The electron images will be recorded with an ultra- stable liquid-helium cooled stage to minimize radiation damage and to optimize electron- optical performance. Distortion correction of the tube images by alignment of successive segments will incorporate a newly developed approach to optimize the extraction of high resolution information, and the computer programs will be automated/made-more-general in collaboration with colleagues working on related specimens at Scripps. Our electron crystallographic methodology provides a unique opportunity to analyze the open ACh receptor channels because it is the only structural method devised so far that can examine this short- lived transition under essentially the same conditions as exist in vivo. It is unlikely that x-ray structures of purified ACh (or other related) receptors, if they are obtained in the future, would be able to deliver such an unambiguous result. PUBLIC HEALTH RELEVANCE: The nicotinic acetylcholine receptor is the best understood member of a family of synaptic ion channels which function in the central and peripheral nervous system, and are pharmaceutical targets for numerous human diseases and psychiatric disorders, including myasthenia gravis, neuromuscular degeneration, epilepsy, depression, nicotine addiction, schizophrenia and Alzheimer's disease. Our structural studies are providing three-dimensional information about the binding sites for anesthetics and other drugs which affect the brain by modulating the function of these receptors. By understanding better the biological mechanisms of these receptors, we contribute basic insight into the nature of the disorders themselves.

描述（由申请人提供）：烟碱乙酰胆碱（ACh）受体在被释放到突触间隙的ACh激活时通过瞬时转变为开放通道形式来介导快速突触传递。我们的目标是了解该事件背后的结构转变，即提供该通道如何工作的明确分子图景。我们研究的富含乙酰胆碱受体的膜来自鱼雷射线的电器官，我们通过冷冻电子显微镜分析了由它们生长的管状膜晶体。拟议的研究建立在长期技术开发计划的基础上，该技术最近产生了闭通道形式的膜结合受体的精细原子模型。为了研究开放通道结构，我们将把含有乙酰胆碱的喷雾液滴推进到位于电磁场上的薄水膜内的管状晶体上。网格，然后通过骤冷捕获反应（在〜2毫秒内）。通过这种方式，我们重现了神经递质与突触受体的短暂反应，并捕获了生理离子环境和天然膜环境中打开的通道。电子图像将使用超稳定的液氦冷却台进行记录，以最大限度地减少辐射损伤并优化电子光学性能。通过连续片段的对齐对管图像进行畸变校正将采用新开发的方法来优化高分辨率信息的提取，并且与斯克里普斯研究相关标本的同事合作，计算机程序将实现自动化/变得更加通用。我们的电子晶体学方法为分析开放的 ACh 受体通道提供了独特的机会，因为它是迄今为止设计的唯一可以在与体内存在的条件基本相同的条件下检查这种短暂转变的结构方法。纯化的乙酰胆碱（或其他相关）受体的 X 射线结构（如果将来获得）不太可能提供如此明确的结果。 公共健康相关性：烟碱乙酰胆碱受体是突触离子通道家族中最了解的成员，在中枢和周围神经系统中发挥作用，并且是许多人类疾病和精神疾病的药物靶标，包括重症肌无力、神经肌肉变性、癫痫、抑郁症、尼古丁成瘾、精神分裂症和阿尔茨海默病。我们的结构研究提供了有关麻醉剂和其他药物的结合位点的三维信息，这些药物通过调节这些受体的功能来影响大脑。通过更好地了解这些受体的生物学机制，我们有助于对疾病本身的本质有基本的了解。