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

项目 1：定位 GABAA 中的全身麻醉结合位点

基本信息

批准号：
8137268
负责人：
JONATHAN Brewer COHEN
金额：
$ 30.26万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8137268
关键词：
Acetylcholine Adverse effects Affinity Agonist Alcohols Amino Acids Anesthetics Barbiturates Binding Binding Sites Brain Cattle Chemical Structure Chemosensitization Cholinergic Receptors Collaborations Cultured Cells Detergents Development Diazomethane Electric Organ Etomidate Family Gated Ion Channel General anesthetic drugs Goals Human Instruction Intravenous Anesthetics Ion Channel Label Location Maps Muscle Neurons Neurotransmitters Nicotinic Receptors Pharmaceutical Preparations Photoaffinity Labels Postsynaptic Membrane Production Propofol Protein Chemistry Proteins Radiolabeled Recombinants Rest Site Skeletal Muscle Structure Synapses Synthesis Chemistry Techniques Testing Time Torpedo Transmembrane Domain analog barbituric acid salt design gamma-Aminobutyric Acid improved inhibitor/antagonist member mutant novel programs radiotracer receptor three dimensional structure

项目摘要

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.

定位 GABAA 和乙酰胆碱受体中的一般麻醉剂结合位点我们的目标是确定 GABAARS（一种临床上使用的一组结构多样的全身麻醉药的主要作用部位，并进行比较它们的结合位点位于烟碱乙酰胆碱受体 (AcChoR) 中，这两个受体都是成员神经递质门控离子通道“半胱环”超家族的成员。大多数麻醉剂都会增强作用 GABA 作用于抑制性 GABAA 受体，同时抑制大脑和骨骼中的兴奋性 AcChoR 肌肉。在单个受体内，口袋中存在多个潜在的麻醉剂结合位点，存在于受体亚基内和亚基界面处，包括离子通道。我们的假设是每种麻醉剂根据结构类别，将优先与不同的受体位点相互作用。一个更好地了解神经递质门控离子内全身麻醉剂结合位点的多样性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和蛋白质化学技术，用于识别光反应性脂肪醇及其类似物的结合位点从牛脑和洗涤剂提取物中分离出的 GABAARS 中的依托咪酯、丙泊酚和巴比妥酸盐培养细胞、从培养细胞中分离出的神经元 a4p2 AcChoR 以及肌肉型 AcChoR 中从鱼雷电器官中分离出烟碱突触后膜。我们将确定是否单个 GABAAR（或 AcChoR）中的麻醉剂结合位点可以被药物占据，根据结构，药物可以发挥作用作为增强剂或抑制剂。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。我们的结构研究是对时间的补充解决了光标记研究（米勒/雷恩斯项目）和突变分析（福尔曼项目）旨在确定我们识别的位点是功能增强还是抑制位点。相关性（参见说明）：大脑中的 GABAARS 是许多临床使用的多种化学麻醉剂的主要作用部位结构，但 GABAARS 内或结构中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。识别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。