General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

描述(申请人提供)：中枢神经系统中的抑制性γ-氨基丁酸A型(GABAA)受体是有效的全身麻醉药依托咪酯、异丙酚、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，增强抑制传递，并降低神经元活性。有关这些药物-受体相互作用的分子结构数据将有助于指导更具选择性、更安全的麻醉药的开发。这项建议的总体目标是更好地确定丙泊酚、依托咪酯、甲氧酮和戊巴比妥在GABAA受体上膜内亚单位-亚单位界面口袋的相互作用。我们打算获得的数据将促进我们对受体通道激活过程中亚单位界面跨膜结构域如何重排、它们所含的麻醉部位的数量以及它们如何与不同的强效麻醉剂相互作用的理解。此外，我们预计将在未来的实验中识别新的GABAA受体突变，旨在确定转基因动物的麻醉机制。我们新的工作假设是，由相邻亚基上的跨膜M1和M3结构域形成的所有五个GABAA受体亚单位界面形成两亲性麻醉剂结合口袋，在离子通道门控过程中改变形状。含有13、21和22个亚基的异五聚体突触GABAA受体形成了四种不同类型的界面袋，可以选择性地与不同的麻醉药相互作用。不同类型的界面袋也可能包含多个不同麻醉药的亚位点，其中一些部分重叠。我们建议对GABAAR11、22和32L亚基之间的跨膜界面进行结构研究，以测试这一假说的关键方面。这些步骤包括：目标1)确定两亲小分子在开放受体和闭合受体中可接触的残基，并确定在这些界面上M1和M3结构域的方向；目标2)识别在没有或存在GABA的情况下影响离子通道开放和关闭的残基；以及目标3)确定这些口袋中影响对强效全身麻醉药作用敏感性的残基，并测试麻醉药和可能的结合决定簇之间的亲和性。我们的实验策略采用扫描半胱氨酸和色氨酸突变体，以及在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巯基特异性化学。电生理数据将使用全球功能变构模型进行分析，并在GABAAR结构的进化同源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改进，保护结果和模型将用于生成新的可测试假说。 公共卫生相关性：在常规临床使用中，全麻药是最有益和最危险的药物之一，但其作用机制仍然知之甚少。需要详细的机制研究来指导改进药物的开发，这些药物可以大幅降低每年数百万患者的麻醉护理的风险和成本。拟议的实验将促进我们对有效的全身麻醉药(依托咪酯、异丙酚、戊巴比妥和阿法西林)在神经系统中的主要分子靶点--伽马氨基丁酸A型受体--的作用位置和方式的理解，通过检验这些药物的分子结合位点是在神经细胞膜内的多个亚单位-亚单位界面(膜内亚单位界面)形成的这一新假说。

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).