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

• 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
• 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

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给药，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全身性疾病的患者。最安全（动物 LD50/ED50 比率最高）的麻醉剂依托咪酯和阿法沙酮都是 GABAA 受体的有效选择性调节剂，并且具有立体选择性。这些药物和类似药物代表了开发改进且更安全的麻醉剂的最佳起点。该更新提案的总体目标是确定依托咪酯、αxalone、丙泊酚（一种强效但安全性较差的麻醉剂）和一种新型强效巴比妥类药物 (mTFD-MPAB) 在五聚体 (α1)2(ß3)2γ2L GABAA 受体位点上的相互作用，并阐明这些位点中将麻醉剂结合与功能效应相结合的构象变化。我们的工作假设是，这些药物作用于由 M1、M2 和 M3 结构域形成的 5 个亚基间跨膜裂隙内的不同位点，并且这些裂隙在受体激活和脱敏过程中扩展。迄今为止，我们的研究已经在多个亚基间裂隙内确定了依托咪酯、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导为增强的受体门控。我们将使用每个亚基跨膜结构域 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，来定义最接近每种麻醉剂的结合裂缝和残基。目标 2 将检验 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，并使用可变大小的突变和半胱氨酸加合物来估计麻醉剂结合袋的有效“截止”大小，以结合在相同的亚基间裂缝中的三种麻醉剂。我们还将使用新型巯基修饰麻醉衍生物和半胱氨酸取代残基来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 受体脱敏率，以及亚基间裂口的形状在开放状态和脱敏状态之间是否不同，同样基于半胱氨​​酸的可及性。这些研究将使用亚毫秒溶液切换和膜片钳电生理学。将使用功能变构模型对数据进行分析，并在不断发展的 GABAAR 结构模型的背景下进一步解释。