Structural Mechanism of the Acetylcholine Receptor in Tubular Membrane Crystals

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中的ACh激活时，通过瞬时转化为开放通道形式介导快速突触传递。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ACh受体丰富的膜来自电鳐的电器官，我们通过冷冻电子显微镜分析从它们生长的管状膜晶体。拟议的研究建立在一个长期的技术发展计划的基础上，该计划最近产生了一个封闭通道形式的膜结合受体的精细原子模型。为了研究开放通道的结构，我们将推进乙酰胆碱含有喷雾液滴的管状晶体躺在一个薄的水膜上的电子显微镜。网格，然后通过骤降冷冻捕获反应（约2 ms内）。通过这种方法，我们概括了神经递质与突触处受体的短暂反应，并在生理离子环境和天然膜环境中捕获开放的通道。电子图像将记录与超稳定的液氦冷却阶段，以尽量减少辐射损伤和优化电子光学性能。通过对齐连续片段对管图像进行失真校正将采用新开发的方法来优化高分辨率信息的提取，并且计算机程序将与Scripps的相关标本的同事合作自动化/更通用。我们的电子晶体学方法提供了分析开放ACh受体通道的独特机会，因为它是迄今为止设计的唯一结构方法，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。纯化的ACh（或其他相关）受体的X射线结构，如果将来获得的话，不太可能提供这样明确的结果。 公共卫生相关性：烟碱乙酰胆碱受体是在中枢和外周神经系统中起作用的突触离子通道家族的最好理解的成员，并且是许多人类疾病和精神障碍（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）的药物靶标。我们的结构研究提供了有关麻醉剂和其他药物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，我们对疾病本身的性质有了基本的了解。

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.