General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

描述（由申请人提供）：广泛使用的全身麻醉剂需要由训练有素和警惕的专家给药，部分原因是它们会产生许多不良反应，并且在临床使用的剂量下可能是致命的，特别是对老年患者和患有全身性疾病的患者。最安全（动物LD50/ED50比率最高）的麻醉药依托咪酯和阿尔法孤独是GABAA受体的有效和选择性调节剂，也显示出立体选择性。这些和类似的药物代表了开发改进的和更安全的麻醉剂的最佳起点。本更新提案的总体目标是确定依托咪酯、阿片类药物、异丙酚（一种强效但不太安全的麻醉剂）和一种新型强效巴比妥酸盐（mTFD-MPAB）在五聚戊二胺（¿1）2（¿3）2？2L GABAA受体，并澄清这些位点的构象变化，将麻醉结合与功能作用结合起来。我们的工作假设是，这些药物在由M1、M2和M3结构域形成的五个亚基跨膜间隙内的不同位点起作用，这些间隙在受体激活和脱敏过程中扩大。到目前为止，我们的研究已经确定了依托咪酯、异丙酚、阿尔法孤单和mTFD-MPAB在多个亚基间隙内的新的接触点。我们还将半胱氨酸改造到这些位点，以揭示gaba增强的巯基修饰和gaba依赖的麻醉保护作用。我们还开发了定量变构模型来解释多种GABAAR突变的功能表型，包括亚位点对每种药物作用的贡献。在目的1中，我们将确定五个亚基间位点中哪些与每种麻醉剂相互作用，哪些亚基将药物结合转化为增强的受体门控。我们将在每个亚基的跨膜结构域M1、M2和M3中使用半胱氨酸取代，以及化学修饰、保护和电生理学，来定义最接近每种麻醉剂的结合间隙和残基。目标2将

DESCRIPTION (provided by applicant): Widely used general anesthetics require administration by highly trained and vigilant experts, in part because they produce many undesirable effects and can be lethal at clinically used doses, particularly in elderly patients an those with systemic diseases. The safest (highest animal LD50/ED50 ratios) anesthetics, etomidate and alphaxalone, are both potent and selective modulators of GABAA receptors that also display stereoselectivity. These and similar drugs represent optimal starting points for developing improved and safer anesthetics. The overall aim of this renewal proposal is to define the interactions of etomidate, alphaxalone, propofol (a potent but less safe anesthetic) and a novel potent barbiturate (mTFD-MPAB) at their sites on pentameric (¿1)2(¿3)2?2L GABAA receptors, and to clarify conformational changes in these sites that couple anesthetic binding to functional effects. Our working hypothesis is that these drugs act at distinct sites within the fiv inter-subunit transmembrane clefts formed by M1, M2, and M3 domains, and that these clefts expand during receptor activation and desensitization. Our studies to date have identified novel contact points for etomidate, propofol, alphaxalone, and mTFD-MPAB within multiple inter-subunit clefts. We also have engineered cysteines into these sites to reveal GABA-enhanced sulfhydryl modification and GABA-dependent anesthetic protection. We have also developed quantitative allosteric models that explain multiple GABAAR mutant functional phenotypes, including subsite contributions to each drug's actions. In Aim 1, we will identify which of the fiv inter-subunit sites interacts with each anesthetic, and which subsites transduce drug binding into enhanced receptor gating. We will use cysteine-substitutions in transmembrane domains M1, M2, and M3 of each subunit, along with chemical modification, protection, and electrophysiology, to define the binding clefts and residues closest to each anesthetic. Aim 2 will test the hypothesis that GABA activation enlarges all five inter-subunit transmembrane clefts in concert. We will examine anesthetic subsite interactions using cysteine accessibility assays and estimate the effective "cut-off" size of the anesthetic binding pockets using variable size mutations and cysteine adducts for three anesthetics that bind in the same inter-subunit clefts. We will also use novel sulfhydryl-modifying anesthetic derivatives and cysteine substituted residues to further define the binding orientation of stereoselective anesthetics. Aim 3 will test whether anesthetics influence GABAA receptor rates of desensitization and whether the shape of the inter-subunit clefts differs between open and desensitized states, again based on cysteine accessibility. These studies will use submillisecond solution switching and patch-clamp electrophysiology. Data will be analyzed using functional allosteric models and further interpreted in the context of evolving GABAAR structural models.