General Anesthetics and nAcCHOR Agonist Affinity

描述(逐字摘录自申请人摘要)。长期的 这个项目的目标是定义分子机制，通过它 全身麻醉药作用于中枢神经系统和外周的蛋白质靶点。这将是 引导开发侧基较少的新型麻醉剂化合物 效果。其总体目标是理清全身麻醉药的作用。 关于激动剂结合、通道门控动力学和激动剂诱导 最佳特征模型配基门控离子通道的脱敏作用 (LGIC)，鱼雷烟碱受体(NAcChoR)，并鉴定 麻醉药物的物理化学特征决定了它们对每一种药物的作用 动力学步长。总体假设是，全身麻醉药作用于 NAcChoR在结构上具有特异性，因为麻醉剂结合亲和力 受到吸引的静电和排斥性位阻的强烈影响 麻醉药与其蛋白质结合部位之间的相互作用。具体的 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增强与该受体上重要功能部位的结合 (2)确定导致nAcChoR通道开放的动力学步骤(S)为 被全麻药改变以确定麻醉剂的分子 体积或化学类别控制着它的作用。 目的2：(1)检验小剂量全身麻醉药增加的假设 与蛋白质结合脱敏的nAcChoR速率常数 严格限制大剂量麻醉药结合的部位和(2)测试 假设全身麻醉药稳定开放通道状态和 通过绑定到相同的小分子来增加脱敏的速率常数 受体结合部位。 拟议的研究将使我们更好地理解 麻醉药与其中枢和外周靶点的相互作用。 选择nAcChoR作为实验模型是因为它的功能远 比任何其他LGIC都定义得更好，允许人们解释 麻醉行动框架内的一个完善和强有力的 动力学模型。用于定义nAcChoR上的麻醉作用的方法是 开发了一种新的快速顺序混合停流荧光分析方法，并 通过可以评估对激动剂结合的麻醉作用的PI来验证， 通道门控和脱敏动力学而不存在潜在的 麻醉剂诱导的通道阻断的混杂作用。

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.