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：
8136477
负责人：
STUART A FORMAN
金额：
$ 36.33万
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 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8136477
关键词：
Affect Agonist Alphaxolone Amino Acids Anesthesia procedures Anesthetics Animals Barbiturates Binding Binding Sites Biological Assay Caring Cell membrane Cells Chemistry Cleaved cell Clinical Cysteine Data Development Disulfides Drug Binding Site Drug Receptors Electrophysiology (science)Epitopes Etomidate Future GABA Agonists GABA-A Receptor General anesthetic drugs Homology Modeling Individual Ion Channel Ion Channel Gating Knock-in Mouse Left Link Maps Measures Mediating Membrane Modeling Modification Molecular Molecular Conformation Molecular Target Muscarinic M1 Receptor Mutagenesis Mutation Nervous system structure Neuraxis Neurons Oocytes Patients Pattern Pentobarbital Pharmaceutical Preparations Propofol Reagent Recombinants Risk Role Scanning Shapes Site Structural Models Structure Synapses System Testing Transgenic Animals Transgenic Organisms Transmembrane Domain Tryptophan Work Xenopus oocyte analog barbituric acid salt base cost crosslink desensitization gamma-Aminobutyric Acid improved interfacial mutant neurosteroids novel public health relevance receptor research study transmission process

项目摘要

DESCRIPTION (provided by applicant): Inhibitory gamma-aminobutyric acid type A (GABAA) receptors in the central nervous system are major targets of the potent general anesthetics etomidate, propofol, barbiturates, and neuroactive steroids. These amphiphilic drugs stabilize conducting GABAA receptor conformations, enhancing inhibitory transmission, and reducing neuronal activity. Molecular level structural data about these drug-receptor interactions will help guide development of more selective, safer anesthetics. The overall aim of this proposal is to better define the interactions of propofol, etomidate, alphaxalone and pentobarbital at intramembrane subunit-subunit interfacial pockets on GABAA receptors. The data we propose to obtain will advance our understanding of how transmembrane domains at subunit interfaces rearrange during receptor channel activation, the number of anesthetic sites they harbor, and how they interact with different potent anesthetics. In addition, we anticipate identifying new GABAA receptor mutations for future experiments aimed at defining anesthetic mechanisms in transgenic animals. Our novel working hypothesis is that all five GABAA receptor subunit interfaces formed by transmembrane M1 and M3 domains on adjacent subunits form amphiphilic anesthetic-binding pockets that change shape during ion channel gating. Hetero-pentameric synaptic GABAA receptors containing 13, 21, and 22 subunits form four distinct types of interfacial pockets that may selectively interact with different anesthetics. Individual types of interfacial pockets also may contain multiple sub-sites for different anesthetics, some partially overlapping. We propose structural studies of the transmembrane interfaces between GABAAR 11, 22, and 32L subunits to test critical aspects of this hypothesis. These include: Aim 1) identifying residues in interfacial pockets that are accessible to small amphiphilic compounds, in open vs. closed receptors, and establishing the orientation of the M1 and M3 domains at these interfaces; Aim2) indentifying residues in these pockets that influence ion channel opening and closing in the absence and presence of GABA; and Aim 3) identifying residues in these pockets that influence sensitivity to the actions of potent general anesthetics, and testing for proximity between anesthetics and putative binding determinants. Our experimental strategy employs scanning cysteine and tryptophan mutants and electrophysiology in recombinant GABAA receptors in two expression systems, and exploits sulfhydryl-specific chemistry in cysteine mutants. Electrophysiological data will be analyzed using global functional allosteric models and further interpreted in the context of evolving homology models of GABAAR structure. Structural models will be refined using cysteine accessibility and both protection results and models will be used to generate new testable hypotheses. PUBLIC HEALTH RELEVANCE: General anesthetics are among the most beneficial and most dangerous drugs in routine clinical use, yet the mechanisms of their actions remain poorly understood. Detailed mechanistic studies are needed to guide development of improved drugs that could substantially reduce both risks and costs of anesthesia care for many millions of patients each year. The proposed experiments will advance our understanding of where and how potent general anesthetics (etomidate, propofol, pentobarbital, and alphaxalone) act on their major molecular targets in the nervous system, gamma-aminobutyric acid type A receptors, by testing the novel hypothesis that molecular binding sites for these drugs are formed at multiple subunit-subunit interfaces within neuronal cell membranes (intra-membrane subunit interfaces).

描述（由申请人提供）：中枢神经系统中A型A型A型抑制性γ-氨基丁酸是有效的全身麻醉剂，丙泊酚，巴贝苯甲酸酯和神经活性类固醇的主要靶标。这些两亲性药物稳定导致GABAA受体构象，增强抑制性传播和降低神经元活性。有关这些药物受体相互作用的分子水平结构数据将有助于指导更具选择性，更安全的麻醉剂的发展。该提案的总体目的是更好地定义Ambrane亚基亚基 - 亚基 - 亚基界面界面口袋在GABAA受体上的相互作用。我们建议获得的数据将促进我们对受体通道激活期间亚基接口的跨膜结构域的理解，它们携带的麻醉位点的数量以及它们如何与不同有效的麻醉药相互作用。此外，我们预计将鉴定出新的GABAA受体突变，以实现旨在定义转基因动物麻醉机制的未来实验。我们新颖的工作假设是，由跨膜M1和M3结构域形成的所有五个GABAA受体亚基界面在相邻亚基上形成了两亲性麻醉结合口袋，它们在离子通道门上改变了形状。包含13、21和22个亚基的杂种五体突触GABAA受体形成了四种不同类型的界面袋，它们可能与不同的麻醉剂相互作用。各种类型的界面口袋也可能包含多个用于不同麻醉药的子站点，有些部分重叠。我们提出了GABAAR 11、22和32L亚基之间跨膜界面的结构研究，以检验该假设的关键方面。其中包括：目标1）识别界面袋中的残基，在开放式和封闭的受体中可以使用的小两亲化合物可进入，并在这些界面处建立M1和M3域的方向； AIM2）在不存在和存在GABA的情况下影响离子通道开口和关闭的这些口袋中的残基；目标3）识别这些口袋中的残留物，从而影响对有效通用麻醉的作用的敏感性，并测试麻醉剂和假定的结合决定因素之间的接近性。我们的实验策略在两个表达系统的重组GABAA受体中采用了扫描半胱氨酸和色氨酸突变体以及电生理学，并利用半胱氨酸突变体中的硫酰基特异性化学性质。电生理数据将使用全球功能变构模型分析，并在Gabaar结构的同源性模型的背景下进一步解释。结构模型将使用半胱氨酸可及性进行完善，保护结果和模型将用于生成新的可检验假设。公共卫生相关性：普通麻醉剂是常规临床用途中最有益，最危险的药物之一，但其行动的机制仍然很少了解。需要详细的机械研究来指导改进的药物的开发，这些药物可以大大降低每年数百万患者的麻醉护理风险和成本。所提出的实验将提高我们对有效的一般麻醉（依托抗，丙泊酚，戊巴比妥和字母酮）在神经系统中的主要分子靶标，γ-氨基丁酸A型受体的主要分子靶标的作用，通过测试这些新型的分子插入这些分子结合位点，以此膜（膜内亚基界面）。