Project 1: Locating General Anesthetic Binding Sites in GABAA & Acetylcholine Rec

GABAA和乙酰胆碱受体中全麻药结合部位的定位 我们的目标是确定GABAARS中全身麻醉药结合位点的数量和位置。 临床使用的结构不同的全麻药组的主要作用部位，并比较 它们与烟碱型乙酰胆碱受体(AcChoRs)的结合部位，这两种受体都是成员 属于神经递质门控离子通道的“Cys-loop”超家族。大多数麻醉剂都能增强这种作用 GABA在抑制GABAA受体的同时抑制脑和骨骼中的兴奋性AcChoRs 肌肉。在单个受体内，口袋中存在多个潜在的麻醉剂结合部位， 存在于受体亚基和亚基界面，包括离子通道。我们的假设是 根据结构类别的不同，每种麻醉剂将优先与不同的受体部位相互作用。一个 提高对神经递质门控离子内全身麻醉剂结合部位多样性的理解 渠道将允许设计更安全的麻醉剂。我们建议使用光亲和标记和 蛋白质化学技术鉴定光反应性脂肪醇及其类似物的结合部位 牛脑洗涤剂提取物中依托咪酯、异丙酚和巴比妥酸盐 培养细胞，从培养细胞分离的神经元型a4p2 AcChoRs和肌型AcChoRs 从鱼雷电器官分离的尼古丁突触后膜。我们将确定是否有一单 GABAAR(或AcChoR)中的麻醉剂结合部位可被药物占据，这些药物根据结构，作用 作为增效剂或抑制剂。这些光亲和标记研究利用了合成化学， 蛋白质化学和蛋白质生产核心。我们的结构研究与时代是相辅相成的- 已解决的光标记研究(Miller/Raines计划)和突变分析(Forman计划) 被设计用来确定我们识别的位置是功能增强还是抑制。 相关性(请参阅说明)： 大脑中的GABA是许多临床上使用的各种化学麻醉剂的主要作用部位 结构，但麻醉剂结合部位的数量和位置在GABA内或在结构上 相关的烟碱型乙酰胆碱受体仍不清楚。确定这些地点将有助于 开发具有更高选择性和更少副作用的麻醉药。

LOCATING GENERAL ANESTHETIC BINDING SITES IN GABAA AND ACETYLCHOLINE RECEPTORS Our goal is to determine the number and location of the binding sites for general anesthetics in GABAARS, a major site of action of a structurally diverse group of general anesthetics used clinically, and to compare them with their binding sites in nicotinic acetylcholine receptors (AcChoRs), receptors that are both members of the "Cys-loop" superfamily of neurotransmitter-gated ion channels. Most anesthetics potentiate the action of GABA at the inhibitory GABAA receptors, while they inhibit the excitatory AcChoRs in brain and skeletal muscle. Within a single receptor multiple potential anesthetic binding sites are present in the pockets that exist within receptor subunits and at subunit interfaces, including the ion channel. It is our hypothesis that each anesthetic, depending on structural class, will interact preferentially with different receptor sites. An improved understanding of the diversity of general anesthetic binding sites within neurotransmitter-gated ion channels will allow for the design of safer anesthetic agents. We propose to use photoaffinity labeling and protein chemistry techniques to identify the binding sites for photoreactive aliphatic alcohols and analogs of etomidate, propofol, and barbiturates in GABAARS isolated from detergent extracts of bovine brain and cultured cells, in neuronal a4p2 AcChoRs isolated from cultured cells, and in muscle-type AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. We will determine whether a single anesthetic binding site in a GABAAR (or AcChoR) can be occupied by drugs that, depending on structure, act as potentiators or inhibitors. These photoaffinity labeling studies make use of the Synthetic Chemistry, Protein Chemistry and Protein Production Cores. Our structural studies are complementary to the time- resolved photolabeling studies in (Miller/Raines Project) and to the mutational analyses (Forman Project) designed to determine whether the sites we identify are sites of functional potentiation or inhibition. RELEVANCE (See instructions): GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine receptors remains unknown. Identification of these sites will contribute to the development of anesthetics with greater selectivity and fewer side-effects.