General Anesthetic Sites on Ligand-Gated Ion Channels

描述(由申请人提供)：在美国，每年约有2500万名患者使用治疗指数非常低的药物进行全身麻醉。全身麻醉的分子机制仍不清楚，这阻碍了改进药物的设计。全身麻醉药作用于配体门控通道的超家族，包括由GABA和甘氨酸门控的抑制性阴离子通道，以及由5-羟色胺和乙酰胆碱门控的兴奋性阳离子通道。这个PPG关注的是 全麻药能够增强抑制性GABA受体(GABAAR)的活性，抑制(并在某些情况下增强)兴奋性神经元5-羟色胺(5-HT3R)和烟碱型乙酰胆碱受体(NAcChoR)。总的假设是，全身麻醉药的不同作用是由这些受体上的一些结合位点介导的，它们的位置和亲和力随麻醉剂的结构和受体的构象而变化，并且两个同源受体之间存在平行关系。PPG的总体目标是：(I)定位GABAA、5-HT3和NACCHO受体上的麻醉剂结合部位，以及(Ii)确定每个部位的功能意义。将使用两种互补的技术来检测位点：光亲和标记(项目2、3和5)和定点突变(项目1、3、4和5)。项目1将描述5-HT3受体的药理学特征，并与项目2和项目3互动，项目2和项目3将确定酒精、依托咪酯和异丙酚光标记的激活(时间分辨光标记)和脱敏受体状态的作用部位。项目4将详细定义使用野生型和突变受体的快速灌流膜片钳技术对GABA受体的麻醉作用的动力学机制，结合光标记结果来指导突变和解释。项目5将使用可光激活的全身麻醉剂来定位GABAA受体上的全身麻醉剂部位。合成化学核心和蛋白质化学核心分别是开发新型光亲和全身麻醉药和定位光掺入部位所必需的。蛋白质生产核心将为每个项目提供神经元受体。

DESCRIPTION (provided by applicant): Some 25 million patients are given general anesthesia each year in the USA using agents with very low therapeutic indices. The molecular mechanisms of general anesthesia remain unknown, hampering the design of improved agents. General anesthetics act on a superfamily of ligand gated channels which include inhibitory anion channels gated by GABA and glycine, and excitatory cation channels gated by serotonin and acetylcholine. This PPG focuses on the ability of general anesthetics to enhance the activity of the inhibitory GABAA receptor (GABAAR) and to inhibit (and in some cases enhance) the excitatory neuronal serotonin (5-HT3R) and nicotinic acetylcholine receptors (nAcChoR). The overall hypothesis is that the various action of general anesthetics are mediated by a number of binding sites on these receptors, that their location and affinity varies with the anesthetic's structure and the receptor's conformation, and that parallels exist between the two homologous receptors. The overall aims of the PPG are to: (i) locate anesthetic binding sites on the GABAA, 5-HT3 and nAcCho receptors, and (ii) define the functional significance of each site. Two complementary techniques will be employed to detect sites, photoaffinity labeling (Projects 2, 3 and 5) and site directed mutagenesis (Projects 1, 3, 4 and 5). Project 1 will characterize the pharmacology of the 5-HT3 receptor and interact with Projects 2 and 3 who will locate the sites of action on activated (time-resolved photolabeling) and desensitized receptor states of alcohols, etomidate and propofol photolabels. Project 4 will define in detail the kinetic mechanisms of anesthetic action on GABAARs using rapid perfusion patch clamp techniques in wild type and mutated receptors, incorporating the photolabeling results to guide mutagenesis and interpretation. Project 5 will locate general anesthetic sites on GABAA receptors using photoactivable general anesthetics. Synthetic and Protein Chemistry Cores are essential for developing novel photoaffinity general anesthetics and for locating the sites of photoincorporation, respectively. A Protein Production Core will supply neuronal receptors to each of the projects.