General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

每年，数以百万计的患者接受警觉的、训练有素的专家的全身麻醉，他们在一定程度上， 管理全身麻醉药物的毒性。根据LD50：ED50比率，最安全的麻醉药是 依托咪酯和安非他酮。两者都主要作为GABAA受体的有效和选择性调节剂， 脑内主要抑制性神经递质门控离子通道。具有协同作用的麻醉剂的组合- Cally on GABAA受体也可能通过减少靶外毒性而提供更大的安全性。长期目标 我们研究的重点是确定全麻药在哪里以及如何作用于GABAA受体 活动。我们之前对这个项目的研究帮助绘制了三组结构不同的跨膜组- 分别与依托咪酯(咪唑)、α-黄体酮(一种神经类固醇)和 立体选择性催眠药巴比妥酸盐R-mTFD-MPAB，我们已经在这些位点上发现了突变 两者都模仿麻醉部位的占据，并阻碍麻醉剂的结合。我们还展示了依托咪酯， 异丙酚和戊巴比妥都作用于GABAA受体，包括突变体，符合简单的两态 变构共激动剂机制。然而，新的初步机制和突变研究表明 类似的模型不能解释字母单独对GABAA受体的作用。此外，mTFD的影响- 突变受体上的MPAB或与野生型受体中依托咪酯的结合表明复杂的相互作用 在GABAA受体上的不同麻醉剂位置之间。我们的新工作假设是 三种部位选择性麻醉药依托咪酯、α-酮和R-mTFD-MPAB对功能有明显影响 受体状态以及这些药物对在GABAA受体和 动物。为了测试这些想法，我们建议使用分子模型、受体突变体、多个电生理- 逻辑方法，机械分析，和一个新的动物模型来评估假说的关键方面。 在目标1中，我们将定义α-黄体酮和内源性神经类固醇THDOC和THDOC的机制 GABAA受体上的别孕酮与依托咪酯受体上的不同。野生型和野生型的机理研究 突变的GABAA受体将在卵母细胞(用于变构模型分析)和HEK293细胞中进行 (用于快速动力学、状态依赖和磷酸化研究)，以解决多种可能性。在目标2中， 我们将量化和比较这三种研究药物组合在野生型GABAA中的效果 并评估和比较每组药物位点突变对所有三种受体作用的影响 麻醉剂。这些研究将利用定量电生理学和我们独特的基于模型的“二元” 变构位移分析。在目标3中，我们将建立配对的药效学效应 一种新型水生动物模型斑马鱼幼体的部位选择性麻醉药。自发性的视频分析 活性和光动力反应将被用来建立等效的药物条件，作为 不同药物对的定量比较及其与目标2结果的相关性。

Millions of patients receive general anesthesia each year from vigilant highly trained experts, who, in part, manage general anesthetic drug toxicities. The safest anesthetic drugs based on LD50:ED50 ratios are etomidate and alphaxalone. Both act primarily as potent and selective modulators of GABAA receptors, the major inhibitory neurotransmitter-gated ion channels in brain. Combinations of anesthetics that act synergisti- cally on GABAA receptors may also provide greater safety by reducing off-target toxicities. The long-term goal of our research is to define both where and how general anesthetics act to modulate GABAA receptor activity. Our previous research for this project has helped map three structurally distinct sets of transmem- brane inter-subunit binding sites for respectively, etomidate (an imidazole), alphaxalone (a neurosteroid), and the stereoselective hypnotic barbiturate R-mTFD-MPAB, and we have identified mutations in these sites that both mimic anesthetic site occupancy and impede anesthetic binding. We also showed that etomidate, propofol, and pentobarbital all act on GABAA receptors, including mutants, in accord with a simple two-state allosteric co-agonist mechanism. However, new preliminary mechanistic and mutant studies indicate that similar models do not account for alphaxalone actions on GABAA receptors. In addition, the effects of mTFD- MPAB on mutant receptors or combined with etomidate in wild-type receptors indicate complex interactions between different sets of anesthetic sites on GABAA receptors. Our new working hypothesis is that the three site-selective anesthetics etomidate, alphaxalone, and R-mTFD-MPAB distinctly affect functional receptor states and that pairs of these drugs differentially synergize in both GABAA receptors and animals. To test these ideas, we propose using molecular models, receptor mutants, multiple electrophysio- logical approaches, mechanistic analysis, and a novel animal model to evaluate key aspects of the hypothesis. In Aim 1, we will define how the mechanisms of alphaxalone and endogenous neurosteroids THDOC and allopregnanolone in GABAA receptors differs from that for etomidate. Mechanistic studies in wild-type and mutant GABAA receptors will be performed in both oocytes (for allosteric model analysis) and HEK293 cells (for rapid kinetic, state-dependence, and phosphorylation studies) to address multiple possibilities. In Aim 2, we will quantify and compare the effects of combining pairs of the three study drugs in wild-type GABAA receptors and assess and compare the effects of mutations in each set of drug sites on actions of all three anesthetics. These studies will utilize quantitative electrophysiology and our unique model-based “binary” allosteric shift analyses. In Aim 3, we will establish the pharmacodynamic effects of combining pairs of the site-selective anesthetics in a novel aquatic animal model, zebrafish larvae. Video analysis of spontaneous activity and photomotor responses will be used to establish equi-effective drug conditions as a basis for quantitative comparison of different drug pairs and correlation with findings from Aim 2.