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

GABAA和乙酰胆碱受体中全身麻醉药结合位点的定位 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， 临床上使用的一组结构不同的全身麻醉药的主要作用部位，并比较 它们与烟碱乙酰胆碱受体（AcChoR）的结合位点，这两种受体都是 神经递质门控离子通道的“半胱氨酸环”超家族。大多数麻醉剂都能增强这种作用 GABA的抑制性GABAA受体，而它们抑制大脑和骨骼肌中的兴奋性AcChoRs 肌肉.在单个受体内，多个潜在的麻醉剂结合位点存在于口袋中， 存在于受体亚单位内和亚单位界面，包括离子通道。我们假设 根据结构类别，每种麻醉剂将优先与不同的受体位点相互作用。一个 提高了对神经递质门控离子通道内全身麻醉剂结合位点多样性的认识 通道将允许设计更安全的麻醉剂。我们建议使用光亲和标记， 蛋白质化学技术，以确定光反应性脂肪醇及其类似物的结合位点， 从牛脑洗涤剂提取物中分离的GABAARS中的依托咪酯、丙泊酚和巴比妥类药物， 在培养的细胞中，在从培养的细胞分离的神经元α 4 β 2 AcChoRs中，以及在培养的细胞中的肌肉型AcChoRs中， 从电鳐电器中分离出的尼古丁突触后膜。我们将决定是否一个单一的 GABAAR（或AcChoR）中的麻醉剂结合位点可以被药物占据，根据结构， 作为增效剂或抑制剂。这些光亲和标记研究利用合成化学， 蛋白质化学和蛋白质生产核心。我们的结构研究是对时间的补充- 解决了（米勒/Raines项目）和突变分析（Forman项目）中的光标记研究 设计来确定我们识别的位点是功能增强还是抑制的位点。 相关性（参见说明）： 脑中的GABA受体是许多临床使用的不同化学物质的麻醉剂的主要作用部位 结构，但在GABAARS或结构中的麻醉剂结合位点的数量和位置 相关的烟碱乙酰胆碱受体仍然未知。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剂。

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.