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然未知，阻碍了改进药物的设计。全身麻醉剂作用于配体门控通道的超家族，包括GABA和甘氨酸门控的抑制性阴离子通道，以及5 -羟色胺和乙酰胆碱门控的兴奋性阳离子通道。本PPG着重于全麻增强抑制性GABAA受体（GABAAR）活性的能力，以及抑制（在某些情况下增强）兴奋性神经元烟碱乙酰胆碱（nAcChoR）和血清素（5HT3R）受体的能力。总的假设是，全身麻醉剂的各种作用是由这些受体上的许多结合位点介导的，它们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)使用麻醉剂光标记定位GABAA、nAcChoR和5-HT3受体上的麻醉剂结合位点，以及（ii）使用快速灌注膜片钳技术确定它们的占据如何变变地调节受体功能。项目1将定位依托咪酯、异丙酚和巴比妥酸盐光标签在GABAAR和nAcChoRs平衡态上的位置。项目2侧重于在门控过程中使用平行电生理和时间分辨光标记的麻醉药与受体的相互作用。项目3将在野生型和突变受体中使用快速灌注膜片钳技术详细定义麻醉作用于GABAARs的动力学机制，并结合光标记结果来指导突变和解释。合成化学核心开发并提供新型光亲和全麻药。蛋白质化学核心定位光结合位点并建立受体同源模型来指导诱变工作。A蛋白生产核心提供大量异种表达，纯化和重组的神经元受体用于光标记。

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.