General Anesthetics and nAcCHOR Agonist Affinity

描述（逐字从申请人的摘要）广泛。长期的 该项目的目标是确定分子机制， 全身麻醉剂作用于CNS和外周中的蛋白质靶。这将 指导开发具有较少副作用的新型麻醉药物 方面的影响.总的目的是解开全身麻醉药的影响 对激动剂结合、通道门控动力学和激动剂诱导的 最佳表征模型配体门控离子通道的脱敏 （LGIC），电鳐烟碱乙酰胆碱受体（nAcChoR），并鉴定 麻醉剂的物理化学特征，这些特征决定了它们对每种 动力学步骤总体假设是全身麻醉药作用于 nAcChoR以结构特异性的方式，因为麻醉剂结合亲和力 受到吸引性静电和排斥性空间的强烈影响 麻醉剂和它们的蛋白质结合位点之间的相互作用。具体 目标是： 目的1：（1）检验静电（偶极，四极， 和/或氢键）之间的相互作用 nAcChoR增强与该受体上功能重要位点的结合， (2)为了鉴定导致nAcChoR通道开放的动力学步骤， 以确定麻醉剂的分子 体积或化学类别决定其作用。 目的2：（1）验证小剂量全身麻醉药增加 nAcChoR通过与蛋白结合而脱敏的速率常数 在空间上限制大麻醉剂结合的位点和（2）测试 假设全身麻醉剂稳定开放通道状态， 通过结合相同的小分子， 受体结合位点 拟议的研究将导致更好地了解基本的 麻醉剂与其CNS和外周靶点之间的相互作用。 选择nAcChoR作为实验模型是因为其功能远 比任何其他LGIC更好地定义，允许一个解释 在一个完善和强大的框架内的麻醉作用 动力学模型用于定义麻醉剂对nAcChoR作用的方法是 开发了一种新的快速顺序混合停流荧光分析方法， 经PI验证，可评估麻醉剂对激动剂结合的作用， 通道门控和脱敏动力学， 麻醉诱导的通道阻滞的混杂效应。

DESCRIPTION (Verbatim from the applicant's abstract) The broad. Iong-term objective of this project is to define the molecular mechanisms by which general anesthetics act on protein targets in the CNS and periphery. This will guide the development of new anesthetic compounds possessing fewer side effects. The overall aim is to disentangle the effects of general anesthetics on agonist binding, channel gating kinetics, and agonist-induced desensitization in the best-characterized model ligand-gated ion channel (LGIC), the Torpedo nicotinic acetyicholine receptor (nAcChoR), and to identify the physicochemical features of anesthetics that govern their action on each kinetic step. The overall hypothesis is that general anesthetics act on the nAcChoR in a structurally specific manner because anesthetic binding affinity is strongly influenced by attractive electrostatic and repulsive steric interactions between anesthetics and their protein binding sites. The specific aims are: Aim 1: (1) to test the hypothesis that electrostatic (dipolar, quadrupolar, and/or hydrogen bonding) interactions between general anesthetics and the nAcChoR enhance binding to functionally important sites on this receptor and (2) to identify the kinetic step(s) leading to nAcChoR channel opening that are altered by general anesthetics to determine whether an anesthetic's molecular volume or chemical class governs its action. Aim 2: (1) to test the hypothesis that small general anesthetics increase nAcChoR's rate constant for desensitization by binding to a protein binding site that sterically limits the binding of large anesthetics and (2) to test the hypothesis that general anesthetics stabilize the open channel state and increase the rate constant for desensitization by binding to the same small receptor binding site. The proposed studies will lead to a better understanding of the fundamental interactions between anesthetics and their targets in the CNS and periphery. The nAcChoR was chosen as the experimental model because its function is far better defined than that of any other LGIC, allowing one to interpret anesthetic actions within the framework of a well-established and robust kinetic model. The method used to define anesthetic actions on the nAcChoR is a new rapid sequential mixing stopped-flow fluorescence assay developed and validated by the PI that can assess anesthetic actions on agonist binding, channel gating, and desensitization kinetics without the potentially confounding effects of anesthetic-induced channel blockade.