General Anesthetic Sites on Ligand-Gated Ion Channels

在美国，每年约有2500万患者使用具有非常低的药物进行全身麻醉。 治疗指标全身麻醉的分子机制仍然未知，阻碍了 更好的代理商全身麻醉剂作用于配体门控通道的超家族，其包括抑制性通道。 由GABA和甘氨酸门控的阴离子通道，以及由5-羟色胺和乙酰胆碱门控的兴奋性阳离子通道。 该PPG关注全身麻醉药增强抑制性GABAA受体活性的能力 （GABAAR），并抑制和在某些情况下增强兴奋性神经元烟碱乙酰胆碱（nAcChoR） 5-羟色胺（5 HT 3R）受体。总的假设是，全身麻醉药的各种作用是 由这些受体上的许多结合位点介导，它们的位置和亲和力随着受体的变化而变化。 麻醉剂的结构和受体的构象。该方案的总体目标是：（一）确定 使用麻醉剂光标记的GABAA、nAcChoR和5-HT 3受体上的麻醉剂结合位点，和（ii） 使用快速灌流膜片钳确定它们的占据如何变构调节受体功能 技术.项目1将确定依托咪酯、丙泊酚和巴比妥酸盐光标记物在平衡状态下的位置， GABAAR和nAcChoRs。项目2的重点是麻醉剂与受体的相互作用，在门控使用在 并行电生理和时间分辨光标记。项目3将详细定义 使用快速灌注膜片钳技术在野生型和 突变的受体，结合光标记结果来指导诱变和解释。合成 化学核心开发和供应新型光亲和性全身麻醉药。蛋白质化学核心 定位光掺入位点，并开发受体的同源模型以指导诱变工作。一 蛋白质生产核心供应大量的异源表达，纯化和重建 用于光标记的神经元受体。

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.