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，部分原因是它们产生许多不良反应，并且在临床使用剂量下可能致命，特别是在老年患者和全身性疾病患者中。最安全的（最高动物LD 50/ED 50比）麻醉剂依托咪酯和阿法沙龙是GABAA受体的强效和选择性调节剂，也显示立体选择性。这些和类似的药物代表了开发改进和更安全的麻醉剂的最佳起点。该更新提案的总体目标是确定依托咪酯、阿法沙龙、丙泊酚（一种强效但安全性较低的麻醉剂）和新型强效巴比妥酸盐（mTFD-MPAB）在五聚体（α1）2（β 3）2γ2L GABAA受体上的相互作用，并阐明这些位点中将麻醉剂结合与功能效应偶联的构象变化。我们的工作假设是，这些药物作用于由M1，M2和M3结构域形成的fiv亚基间跨膜裂缝内的不同位点，并且这些裂缝在受体活化和脱敏期间扩大。迄今为止，我们的研究已经确定了依托咪酯、丙泊酚、阿法沙龙和mTFD-MPAB在多个亚基间裂隙内的新接触点。我们还将半胱氨酸工程化到这些位点中，以揭示GABA增强的巯基修饰和GABA依赖的麻醉保护。我们还开发了定量变构模型，解释多种GABAAR突变体的功能表型，包括每个药物的作用亚位点的贡献。在目的1中，我们将确定哪些fiv亚基间位点与每种麻醉剂相互作用，哪些亚位点使药物结合增强受体门控。我们将使用每个亚基的跨膜结构域M1、M2和M3中的半胱氨酸取代，沿着化学修饰、保护和电生理学，以确定最接近每种麻醉剂的结合裂缝和残基。目的2将测试的假设，GABA激活扩大所有五个亚基间跨膜裂缝在音乐会。我们将研究麻醉剂亚位点的相互作用，使用半胱氨酸可及性测定和估计的有效“截止”的麻醉剂结合口袋的大小，使用可变大小的突变和半胱氨酸加合物的三种麻醉剂，结合在相同的亚基间裂缝。我们还将使用新型巯基修饰麻醉剂衍生物和半胱氨酸取代的残基来进一步定义立体选择性麻醉剂的结合方向。目的3将测试麻醉剂是否影响GABAA受体脱敏率，以及亚基间裂缝的形状在开放和脱敏状态之间是否不同，再次基于半胱氨酸可及性。这些研究将使用亚毫秒溶液转换和膜片钳电生理学。数据将使用功能变构模型进行分析，并在不断发展的GABAAR结构模型的背景下进一步解释。

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 (α1)2(ß3)2γ2L 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.