General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

GABA-A 受体亚基界面袋中的全身麻醉位点

基本信息

批准号：
9312844
负责人：
STUART A FORMAN
金额：
$ 33.59万
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 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9312844
关键词：
Affect Affinity Agonist Alphaxolone Amino Acids Anesthetics Animals Barbiturates Binding Biological Assay Chemicals Clinical Cysteine Dangerousness Data Dental Cavity Lining Development Dose Drug effect disorder Electrophysiology (science)Engineering Etomidate Future GABA-A Receptor General anesthetic drugs Grant Homology Modeling Hydrophobicity Intravenous Anesthetics Investigation Lethal Dose 50 Maps Measures Mediating Modeling Modification Molecular Molecular Conformation Mutation Nature Pharmaceutical Preparations Phenotype Process Propofol Publishing Reagent Receptor Activation Recovery Reporting Rest Series Shapes Site Steroids Structural Models Synapses Systemic disease Techniques Testing Time Training Transmembrane Domain Work Xenopus oocyte adduct amphiphilicity base desensitization experimental study gamma-Aminobutyric Acid improved insight interfacial methanethiosulfonate mutant neurotransmission novel older patient patch clamp public health relevance receptor theories

项目摘要

DESCRIPTION (provided by applicant): Widely used general anesthetics require administration by highly trained and vigilant experts, in part because they produce many undesirable effects and can be lethal at clinically used doses, particularly in elderly patients an those with systemic diseases. The safest (highest animal LD50/ED50 ratios) anesthetics, etomidate and alphaxalone, are both potent and selective modulators of GABAA receptors that also display stereoselectivity. These and similar drugs represent optimal starting points for developing improved and safer anesthetics. The overall aim of this renewal proposal is to define the interactions of etomidate, alphaxalone, propofol (a potent but less safe anesthetic) and a novel potent barbiturate (mTFD-MPAB) at their sites on pentameric (α1)2(ß3)2γ2L GABAA receptors, and to clarify conformational changes in these sites that couple anesthetic binding to functional effects. Our working hypothesis is that these drugs act at distinct sites within the fiv inter-subunit transmembrane clefts formed by M1, M2, and M3 domains, and that these clefts expand during receptor activation and desensitization. Our studies to date have identified novel contact points for etomidate, propofol, alphaxalone, and mTFD-MPAB within multiple inter-subunit clefts. We also have engineered cysteines into these sites to reveal GABA-enhanced sulfhydryl modification and GABA-dependent anesthetic protection. We have also developed quantitative allosteric models that explain multiple GABAAR mutant functional phenotypes, including subsite contributions to each drug's actions. In Aim 1, we will identify which of the fiv inter-subunit sites interacts with each anesthetic, and which subsites transduce drug binding into enhanced receptor gating. We will use cysteine-substitutions in transmembrane domains M1, M2, and M3 of each subunit, along with chemical modification, protection, and electrophysiology, to define the binding clefts and residues closest to each anesthetic. Aim 2 will test the hypothesis that GABA activation enlarges all five inter-subunit transmembrane clefts in concert. We will examine anesthetic subsite interactions using cysteine accessibility assays and estimate the effective "cut-off" size of the anesthetic binding pockets using variable size mutations and cysteine adducts for three anesthetics that bind in the same inter-subunit clefts. We will also use novel sulfhydryl-modifying anesthetic derivatives and cysteine substituted residues to further define the binding orientation of stereoselective anesthetics. Aim 3 will test whether anesthetics influence GABAA receptor rates of desensitization and whether the shape of the inter-subunit clefts differs between open and desensitized states, again based on cysteine accessibility. These studies will use submillisecond solution switching and patch-clamp electrophysiology. Data will be analyzed using functional allosteric models and further interpreted in the context of evolving GABAAR structural models.

描述（由申请人提供）：广泛使用的全身麻醉剂需要由训练有素且警惕的专家进行管理，部分原因是它们会产生许多不良反应，并且在临床使用剂量下可能致命，特别是对于老年患者和患有最高动物 LD50 的患者。 /ED50 比率）麻醉剂依托咪酯和阿法沙酮都是 GABAA 受体的有效和选择性调节剂，也表现出立体选择性。该更新提案的总体目标是确定依托咪酯、阿法沙酮、丙泊酚（一种强效但安全性较低的麻醉剂）和一种新型强效巴比妥类药物（mTFD-MPAB）在其部位的相互作用。五聚体 (α1)2(ß3)2γ2L GABAA 受体，并阐明这些位点的构象变化，这些位点将麻醉剂结合与功能作用结合起来，我们的工作假设是这些药物作用于不同的位点。在由 M1、M2 和 M3 结构域形成的 5 个亚基间跨膜裂隙内，这些裂隙在受体激活和脱敏过程中扩大，迄今为止，我们的研究已经确定了依托咪酯、丙泊酚、alphaxalone 和 mTFD-MPAB 的新接触点。我们还将半胱氨酸改造到这些位点，以揭示 GABA 增强的巯基修饰和 GABA 依赖性麻醉保护。我们还开发了定量变构模型来解释多种 GABAAR 突变体功能表型，包括亚位点对每种药物作用的贡献。在目标 1 中，我们将确定五个亚基间位点中的哪一个与每种麻醉剂相互作用，以及哪些亚位点将药物结合转变成。我们将使用每个亚基跨膜域 M1、M2 和 M3 中的半胱氨酸取代，以及化学修饰、保护和电生理学来定义结合。目标 2 将测试 GABA 激活同时扩大所有五个亚基间跨膜裂隙的假设，我们将使用半胱氨酸可及性测定来检查麻醉亚位点相互作用，并估计有效的“截止”大小。使用不同大小的突变和半胱氨酸加合物对麻醉剂结合袋进行结合，以结合在相同的亚基间裂缝中。我们还将使用新型的巯基修饰麻醉剂。衍生物和半胱氨酸取代残基进一步定义立体选择性麻醉剂的结合方向，目标 3 将测试麻醉剂是否影响 GABAA 受体的脱敏率，以及开放状态和脱敏状态之间亚基间裂缝的形状是否不同，同样基于半胱氨酸。这些研究将使用亚毫秒溶液切换和膜片钳电生理学，并使用功能变构模型对数据进行分析，并在不断发展的背景下进行进一步解释。 GABAAR结构模型。