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: 8299577
• 负责人: STUART A FORMAN
• 金额: $ 35.98万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299577
• 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

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（由申请方提供）：中枢神经系统中的抑制性γ-氨基丁酸A型（GABAA）受体是强效全身麻醉药依托咪酯、丙泊酚、巴比妥酸盐和神经活性类固醇的主要靶点。这些两亲性药物稳定传导GABAA受体构象，增强抑制性传递，并降低神经元活性。这些药物-受体相互作用的分子水平的结构数据将有助于指导更有选择性，更安全的麻醉剂的发展。本提案的总体目标是更好地确定丙泊酚、依托咪酯、阿法沙龙和戊巴比妥在GABAA受体的膜内亚基-亚基界面口袋中的相互作用。我们建议获得的数据将推进我们的理解，如何跨膜结构域在亚基接口在受体通道激活过程中重新排列，麻醉位点的数量，他们港，以及他们如何与不同的强效麻醉剂相互作用。此外，我们预计确定新的GABAA受体突变的未来实验，旨在定义在转基因动物的麻醉机制。我们新的工作假设是，所有五个GABAA受体亚基的跨膜M1和M3结构域相邻的亚基上形成的接口形成两亲性的麻醉剂结合口袋，改变形状在离子通道门控。含有13、21和22个亚基的异源五聚体突触GABAA受体形成四种不同类型的界面口袋，它们可以选择性地与不同的麻醉剂相互作用。个别类型的界面袋也可能包含用于不同麻醉剂的多个子部位，一些部分重叠。我们建议GABAAR 11，22和32 L亚基之间的跨膜界面的结构研究，以测试这一假设的关键方面。这些措施包括：目的1）鉴定在开放与封闭受体中，小的两亲性化合物可接近的界面口袋中的残基，并建立M1和M3结构域在这些界面处的取向;目的2）鉴定这些口袋中在GABA不存在和存在下影响离子通道打开和关闭的残基;和目的3）鉴定这些口袋中影响对强效全身麻醉剂作用的敏感性的残基，并测试麻醉剂和推定的结合决定簇之间的接近性。我们的实验策略采用扫描半胱氨酸和色氨酸突变体和电生理学在重组GABAA受体在两个表达系统，并利用巯基特异性化学半胱氨酸突变体。电生理数据将使用全球功能变构模型进行分析，并进一步解释GABAAR结构的同源性模型的发展。结构模型将使用半胱氨酸可及性进行改进，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：全身麻醉药是常规临床使用中最有益和最危险的药物之一，但其作用机制仍知之甚少。需要详细的机制研究来指导改进药物的开发，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。拟议的实验将推进我们的理解，在哪里以及如何有效的全身麻醉剂（依托咪酯，丙泊酚，戊巴比妥，和阿法沙龙）作用于他们的主要分子靶点在神经系统中，γ-氨基丁酸A型受体，通过测试的新假设，这些药物的分子结合位点形成在多个亚基-亚基界面内神经元细胞膜（膜内亚基界面）。