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，其中包括由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。这种PPG集中于全麻药增强抑制性GABA受体(GABAAR)的活性以及抑制和在某些情况下增强兴奋性神经元烟碱乙酰胆碱(NAcChoR)和5-羟色胺(5HT3R)受体的能力。总的假设是，全身麻醉药的各种作用是由这些受体上的一些结合位点介导的，这些结合位点的位置和亲和力随着麻醉剂的结构和受体的构象而变化。PPG的总体目标是：(I)使用麻醉光标记法定位GABAA、nAcChoR和5-HT3受体上的麻醉剂结合位点，以及(Ii)利用快速灌流膜片钳技术确定它们的占位如何以变构方式调节受体功能。项目1将在GABAAR和nAcChoRs的平衡态上定位依托咪酯、异丙酚和巴比妥酸盐光标记的位置。项目2侧重于在并行电生理和时间分辨光标记中，在门控过程中麻醉剂与受体的相互作用。项目3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。合成化学核心公司开发并提供新型光亲和型全身麻醉药。蛋白质化学核心定位光结合的位置，并开发受体的同源模型来指导突变工作。蛋白质生产核心为光标记提供大量异源表达、纯化和重组的神经元受体。

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 nicotinic acetylcholine (nAcChoR) and serotonin (5HT3R) receptors. The overall hypothesis is that the various actions 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. The overall Aims of the PPG are to: (i) locate the anesthetic binding sites on the GABAA, nAcChoR and 5-HT3 receptors using anesthetic photolabels, and (ii) define how their occupancy allosterically modulates receptor function using rapid perfusion patch clamp techniques. Project 1 will locate sites of etomidate, propofol and barbiturate photolabels on equilibrium states of GABAAR and nAcChoRs. Project 2 focuses on the interaction of anesthetics with receptors during gating using in parallel electrophysiological and time-resolved photolabeling. Project 3 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. A Synthetic Chemistry Core develops and supplies novel photoaffinity general anesthetics. A Protein Chemistry Core locates the sites of photoincorporation and develops homology models of receptors to guide mutagenesis work. A Protein Production Core supplies large quantities of heterologously expressed, purified and reconstituted neuronal receptors for photolabeling.