General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

描述（由申请人提供）：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、异丙酚、巴比妥类和神经活性类固醇的主要靶标。这些两亲性药物可稳定传导 GABAA 受体构象，增强抑制性传递并减少神经元活动。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开发更具选择性、更安全的麻醉剂。该提案的总体目标是更好地定义丙泊酚、依托咪酯、阿法沙酮和戊巴比妥在 GABAA 受体膜内亚基-亚基界面袋处的相互作用。我们打算获得的数据将加深我们对受体通道激活过程中亚基界面跨膜结构域如何重新排列、它们所具有的麻醉位点数量以及它们如何与不同强效麻醉剂相互作用的理解。此外，我们预计会为未来旨在定义转基因动物麻醉机制的实验确定新的 GABAA 受体突变。我们的新工作假设是，所有五个 GABAA 受体亚基界面由相邻亚基上的跨膜 M1 和 M3 结构域形成，形成两亲性麻醉剂结合袋，这些袋在离子通道门控期间改变形状。含有 13、21 和 22 个亚基的异五聚体突触 GABAA 受体形成四种不同类型的界面袋，可以选择性地与不同的麻醉剂相互作用。各种类型的界面袋还可能包含用于不同麻醉剂的多个子位点，其中一些子位点部分重叠。我们建议对 GABAAR 11、22 和 32L 亚基之间的跨膜界面进行结构研究，以测试该假设的关键方面。这些包括： 目标 1) 识别开放与封闭受体中小两亲性化合物可接近的界面袋中的残基，并确定这些界面处 M1 和 M3 结构域的方向；目标2) 识别这些口袋中在 GABA 不存在和存在的情况下影响离子通道打开和关闭的残基；目标 3) 识别这些口袋中影响强效全身麻醉药作用敏感性的残留物，并测试麻醉药和假定的结合决定因素之间的接近度。我们的实验策略采用扫描半胱氨酸和色氨酸突变体以及两个表达系统中重组 GABAA 受体的电生理学，并利用半胱氨酸突变体中的硫氢基特异性化学。电生理数据将使用全局功能变构模型进行分析，并在 GABAAR 结构进化同源模型的背景下进一步解释。将使用半胱氨酸可及性来完善结构模型，并且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：全身麻醉剂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。所提出的实验将通过测试这些药物的分子结合位点在神经元细胞膜内的多个亚基-亚基界面（膜内亚基界面）上形成的新假设，将加深我们对强效全身麻醉剂（依托咪酯、丙泊酚、戊巴比妥和阿法沙酮）在神经系统中的主要分子靶点——γ-氨基丁酸A型受体上作用的位置和方式的理解。

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).