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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

基本信息

批准号：
8636024
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
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-01 至 2016-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8636024
关键词：
Acetylcholine Affect Alzheimer&apos 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 public health relevance receptor receptor binding receptor function user-friendly

项目摘要

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.

描述(申请人提供)：烟碱型乙酰胆碱(ACh)受体被释放到突触间隙的ACh激活时，瞬间转换为开放通道形式，从而介导快速突触传递。我们的目标是了解这一事件背后的结构转变，即提供通道如何工作的明确分子图像。我们研究的富含ACh受体的膜来自于鱼雷射线的电器官，我们用冷冻电子显微镜分析了从它们生长出来的管状膜晶体。这项拟议的研究建立在长期技术开发计划的基础上，最近产生了闭合通道形式的膜结合受体的精炼原子模型。为了研究开放的通道结构，我们将把含有乙酰胆碱的喷雾液滴推进到位于电磁显微镜上的薄水膜中的管状晶体上。网格，然后通过骤降冷冻捕获反应(约2ms内)。通过这种方法，我们概括了神经递质与突触上的受体的短暂反应，并在生理离子环境和天然膜环境中捕获了开放的通道。电子图像将使用超稳定的液氦冷却阶段进行记录，以将辐射损害降至最低，并优化电子光学性能。通过对连续片段进行排列来校正管子图像的失真将采用一种新开发的方法来优化高分辨率信息的提取，并且计算机程序将与斯克里普斯研究所从事相关标本工作的同事合作实现自动化/变得更加通用。我们的电子结晶学方法学为分析开放的ACh受体通道提供了一个独特的机会，因为它是迄今为止唯一设计的结构方法，可以在与体内存在的基本相同的条件下检查这种短暂的转变。如果在未来获得纯化的ACh(或其他相关的)受体的X射线结构，它不太可能能够提供如此明确的结果。