Structural Mechanism of the Acetylcholine Receptor in Tubular Membrane Crystals

描述（由申请人提供）：当释放到突触间隙的乙酰胆碱激活时，烟碱乙酰胆碱（ACh）受体通过瞬间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。我们研究的富含乙酰胆碱受体的膜来自鱼雷射线的电器官，我们用低温电子显微镜分析了从中生长出来的管状膜晶体。这项提议的研究建立在一个长期的技术发展计划上，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。为了研究开放通道结构，我们将把含有乙酰胆碱的喷雾液滴推进到放置在薄膜上的管状晶体上，然后通过暴跌冷冻（在~2ms内）捕获反应。通过这种方法，我们概括了神经递质与突触受体的简短反应，并捕获了生理离子环境和天然膜环境下开放的通道。电子图像将被记录在一个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。通过对连续片段的对齐来校正试管图像的畸变，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相关标本的同事合作，实现自动化/更通用。我们的电子晶体学方法为分析开放的乙酰胆碱受体通道提供了一个独特的机会，因为它是迄今为止设计的唯一的结构方法，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。纯化乙酰胆碱（或其他相关的）受体的x射线结构，如果将来获得的话，不太可能提供如此明确的结果。

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.