GENERAL ANESTHETIC SITES ON LIGAND GATED ION CHANNELS

每年约有 2500 万患者接受全身麻醉 美国使用治疗指数非常低的药物。分子 全身麻醉的机制仍然未知，阻碍了设计 改进的代理。据信全身麻醉剂可以调节功能 突触后配体门控离子通道同源超家族的成员。 该 PPG 专注于增强抑制性 GABA/A 受体的作用 （GABAAR）和对兴奋性烟碱受体的抑制作用， nAcChoR。总体假设是全身麻醉剂与 这些受体上的位点数量，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， 两个同源受体之间存在相似之处。整体 PPG 的目标是： (i) 在 GABA/A 上定位麻醉部位，并且 nAcCho 受体，以及 (ii) 定义其功能意义。焦点 位于受体的三个区域：细胞外部分， 特别是在拳头跨膜螺旋之前，第二个 跨膜螺旋和脂质蛋白界面。两个互补 将采用技术来检测位点、光亲和标记 （项目 I、II 和 IV）和定点诱变（项目 III 和 IV）。 项目二将定位挥发性物质、酒精和类固醇的场所 麻醉剂对 nAcChoR 的平衡状态进行照片标记，并进行项目 IV 将使用类似的技术以及定点诱变， 找到 GABAAR 上的站点。项目我将确定哪些位点抑制 nAcChoR 的开放通道使用时间分辨照片标记。项目三 将使用快速灌注膜片钳从动力学上定义机制 野生型和突变受体的技术，结合了光- 标记结果以指导诱变和解释。项目一和二 将研究胆固醇位点在调节变构中的作用 麻醉部位和类固醇麻醉作用之间的相互作用， 分别。合成和蛋白质化学核心对于 开发新型光亲和全身麻醉剂并定位 分别是光掺入位点。

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 are believed to modulate the function of a homologous super-family of postsynaptic ligand-gated ion channels. This PPG focuses on the enhancing action on the inhibitory GABA/A receptor (GABAAR) and the inhibitory action on the excitatory nicotinic receptor, nAcChoR. The overall hypothesis is that general anesthetics bind to a number of 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 anesthetics sites on the GABA/A and nAcCho receptors, and (ii) define their functional significance. The focus is on three regions of the receptors: the extracellular portion, particularly just before the fist transmembrane helix, the second transmembrane helix, and the lipid protein interface. Two complementary techniques will be employed to detect sites, photoaffinity labeling (Projects I, II & IV) and site directed mutagenesis (Projects III and IV). Project II will locate the sites where volatile, alcohol and steroid anesthetics photo-label the equilibrium states of the nAcChoR, and Project IV will use similar techniques, as well as site directed mutagenesis, to locate sites on the GABAAR. Project I will determine which sites inhibit the nAcChoR's open channel using time resolved photo-labeling. Project III will define mechanisms kinetically using rapid perfusion patch clamp techniques in wild type and mutated receptors, incorporating the photo- labeling results to guide mutagenesis and interpretation. Projects I & II will investigate the role of cholesterol sites in modulating allosteric interactions between anesthetic sites and steroid anesthetic action, respectively. Synthetic and Protein Chemistry Cores are essential for developing novel photoaffinity general anesthetics and for locating the sites of photo-incorporation, respectively.