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剂依托咪酯、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，降低神经元活性。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有选择性、更安全的麻醉剂的开发。本提案的总体目的是更好地定义丙泊酚、依托咪酯、阿尔法孤单和戊巴比妥在GABAA受体的膜内亚基-亚基界面口袋中的相互作用。我们拟获得的数据将有助于我们理解亚基界面的跨膜结构域在受体通道激活过程中如何重排，它们所包含的麻醉位点的数量，以及它们如何与不同的强效麻醉剂相互作用。此外，我们期望在未来的实验中发现新的GABAA受体突变，旨在确定转基因动物的麻醉机制。我们的新工作假设是，相邻亚基上的跨膜M1和M3结构域形成的所有五个GABAA受体亚基界面形成两亲性麻醉结合口袋，这些口袋在离子通道门控过程中改变形状。含有13、21和22亚基的异五聚体突触GABAA受体形成四种不同类型的界面口袋，可以选择性地与不同的麻醉剂相互作用。个别类型的界面口袋也可能包含不同麻醉剂的多个子位点，有些部分重叠。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，以检验这一假设的关键方面。这些包括：目标1)识别小的两亲性化合物在开放和封闭受体中可接近的界面口袋中的残基，并确定这些界面上M1和M3结构域的方向；目的2)识别在GABA缺失和存在的情况下影响离子通道打开和关闭的这些口袋中的残基；目标3)识别这些口袋中影响对强效全身麻醉药作用敏感性的残留物，并测试麻醉药与推定的结合决定因素之间的接近性。我们的实验策略采用扫描半胱氨酸和色氨酸突变体以及在两种表达系统中重组GABAA受体的电生理学，并利用半胱氨酸突变体的巯基特异性化学。电生理数据将使用全局功能变构模型进行分析，并在不断发展的GABAAR结构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来改进，保护结果和模型将用于产生新的可测试的假设。

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).