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: 8775923
• 负责人: STUART A FORMAN
• 金额: $ 34.75万
• 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/8775923
• 关键词: Affect; Affinity; Agonist; Alphaxolone; Amino Acids; Anesthetics; Animals; Barbiturates; Binding; Biological Assay; Chemicals; Cleaved cell; Clinical; Cysteine; DNA Sequence Rearrangement; Data; Dental Cavity Lining; Development; Dose; Drug effect disorder; Electrophysiology (science); Engineering; Etomidate; Future; GABA-A Receptor; General anesthetic drugs; Grant; Homology Modeling; Intravenous Anesthetics; Investigation; Lethal Dose 50; Maps; Measures; Mediating; Modeling; Modification; Molecular; Mutation; Nature; Pharmaceutical Preparations; Phenotype; Process; Propofol; Publishing; Reagent; Receptor Activation; Recovery; Reporting; Rest; Series; Shapes; Site; Solutions; Steroids; Structural Models; Synapses; Systemic disease; Techniques; Testing; Time; Training; Transmembrane Domain; Work; Xenopus oocyte; adduct; barbituric acid salt; base; chemical substitution; desensitization; gamma-Aminobutyric Acid; improved; insight; interfacial; methanethiosulfonate; mutant; neurotransmission; novel; older patient; patch clamp; public health relevance; receptor; research study; 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 (¿1)2(¿3)2?2L 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(申请人提供)：广泛使用的全身麻醉药需要由训练有素且警惕的专家给药，部分原因是它们会产生许多不良影响，在临床使用剂量时可能是致命的，特别是在老年患者和有全身疾病的患者中。最安全的麻醉药(动物LD50/ED50比率最高)是依托咪酯和α-黄体酮，它们都是GABAA受体的有效和选择性调节剂，也表现出立体选择性。这些药物和类似的药物是开发改进和更安全的麻醉药的最佳起点。这项更新建议的总体目标是确定依托咪酯、安非他明、异丙酚(一种有效但安全性较低的麻醉剂)和新的有效巴比妥酸盐(mTFD-MPAB)在五聚体(1)2(3)2？2L GABAA受体上的相互作用，并阐明这些位点的构象变化将麻醉剂结合到功能效应上。我们的工作假设是，这些药物作用于由M1、M2和M3结构域形成的五个亚单位间跨膜裂隙中的不同位置，这些裂隙在受体激活和脱敏过程中扩大。到目前为止，我们的研究已经确定了依托咪酯、异丙酚、α-单抗和mTFD-MPAB在多个亚基间裂隙中的新接触点。我们还将半胱氨酸改造到这些部位，以揭示GABA增强的巯基修饰和GABA依赖的麻醉剂保护。我们还开发了解释多种GABAAR突变功能表型的定量变构模型，包括对每种药物作用的亚位点贡献。在目标1中，我们将确定哪五个亚基间位点与每种麻醉剂相互作用，以及哪些亚基将药物结合转导到增强的受体门控。我们将使用每个亚基跨膜结构域M1、M2和M3上的半胱氨酸取代，以及化学修饰、保护和电生理学，来定义与每个麻醉剂最接近的结合裂隙和残基。目标2将 验证这样一种假设，即GABA的激活会同时扩大所有五个亚基间跨膜裂隙。我们将使用半胱氨酸可及性分析来检查麻醉剂亚基之间的相互作用，并使用可变大小的突变和三种结合在相同亚基间裂隙中的麻醉药的半胱氨酸加合物来估计麻醉剂结合口袋的有效“截止”大小。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来进一步确定立体选择性麻醉剂的结合方向。目的3将测试麻醉药是否影响GABAA受体的脱敏速率，以及亚基间裂隙的形状在开放和脱敏状态下是否不同，同样是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。数据将使用功能变构模型进行分析，并在不断演变的GABAAR结构模型的背景下进一步解释。