3D Structure and Function of Neuronal Nicotinic Acetylcholine Receptors

描述（由申请人提供）： 神经元烟碱乙酰胆碱受体（nAChR）是介导快速突触传递的五聚体配体门控离子通道，并且是神经退行性疾病和精神疾病的重要和新颖的治疗靶点。目前还没有任何烟碱受体的原子分辨率结构，并且在这个Cys环受体超家族中没有受体在结构上具有一种以上的构象。该研究的长期目标是更好地了解不同功能状态之间转换的基础构象变化，并开发与结构导向药物设计相关的可靠的nAChR原子分辨率模型。为实现这些目标，将采用以下方法。1.通过受体直向同源物筛选，鉴定用于结晶的α 7受体候选物，并测试条件以优化受体在洗涤剂中的稳定性。2.使用快速灌注膜片钳和单通道分析，测试结构修改和药理学探针的组合，以稳定不同的受体构象。3.使不同构象状态的受体结晶，并确定（a）封闭静息受体、（B）开放激活受体和（c）封闭脱敏受体的原子分辨率结构。结构研究将与功能测定相补充，功能测定测试关于受体状态转换机制的新的基于结构的假设。这些结果将对Cys环受体家族和α 7 nAChRs产生广泛的影响。 公共卫生相关性： 拟议的研究将提供第一个可靠的结构模板，用于设计对阿尔茨海默病和帕金森病以及精神分裂症有用的高度选择性和创新性疗法。此外，了解这类重要蛋白质的结构如何在激活后发生变化，将有助于深入了解生物物理过程 对成瘾和其他人类神经系统健康问题非常重要。

DESCRIPTION (provided by applicant): Neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that mediate fast synaptic transmission and are important and novel therapeutic targets in neurodegenerative diseases and mental illness. Currently there is no atomic-resolution structure for any nicotinic receptor, and no receptor in this Cys-loop receptor superfamily has been structurally characterized in more than one conformation. The long-term objectives of the research are to better understand the conformational changes that underlie the transitions between different functional states and to develop reliable, atomic-resolution models of nAChRs relevant to structure-guided drug design. Toward these goals the following approach will be used. 1. Through receptor ortholog screening, identify an alpha7 receptor candidate for crystallization, and test conditions to optimize receptor stability in detergent. 2. Using fast-perfusion patch clamp and single channel analysis, test combinations of construct modifications and pharmacological probes to stabilize distinct receptor conformations. 3. Crystallize receptor in different conformational states and determine atomic-resolution structures of (a) a closed-resting receptor, (b) an open-activated receptor and (c) a closed-desensitized receptor. The structural studies will be complemented with functional assays that test new structure-based hypotheses regarding mechanisms of state transitions in the receptor. The results will have broad implications for the Cys-loop receptor family in general and alpha7 nAChRs specifically. PUBLIC HEALTH RELEVANCE: The proposed studies will provide the first reliable structural templates for design of highly selective and innovative therapeutics useful in Alzheimer's and Parkinson's diseases and schizophrenia. Moreover, an understanding of how the structure of this important class of proteins changes upon activation will lend insight into biophysical process of great importance in addiction and other human nervous system health issues.