Structural Mechanism of the Acetylcholine Receptor in Tubular Membrane Crystals

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。 与公共健康相关：烟碱型乙酰胆碱受体是在中枢和外周神经系统发挥作用的突触离子通道家族中最被了解的成员，是许多人类疾病和精神障碍的药物靶点，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。我们的结构研究正在提供有关麻醉剂和其他药物的结合部位的三维信息，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做出了基本的见解。

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.