3D Structure and Function of Neuronal Nicotinic Acetylcholine Receptors

描述（申请人提供）：神经元烟碱乙酰胆碱受体（nAChRs）是介导快速突触传递的五聚体配体门控离子通道，是神经退行性疾病和精神疾病的重要和新的治疗靶点。目前还没有任何尼古丁受体的原子分辨结构，并且在这个Cys-loop受体超家族中没有一个受体具有多个构象的结构特征。该研究的长期目标是更好地理解不同功能状态之间转变的构象变化，并开发与结构引导药物设计相关的可靠的原子分辨率nachr模型。为了实现这些目标，将使用以下方法。1. 通过受体同源筛选，确定α 7受体的结晶候选物，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，测试结构修饰和药理学探针的组合，以稳定不同的受体构象。3. 结晶不同构象状态的受体，并确定(a)封闭静止受体，(b)开放激活受体和(c)封闭脱敏受体的原子分辨率结构。结构研究将辅以功能分析，以测试关于受体状态转变机制的新的基于结构的假设。该结果将对Cys-loop受体家族，特别是alpha7 nachr具有广泛的意义。

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.