General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

每年有数百万患者接受由训练有素的专家进行的全身麻醉， 管理全身麻醉药物毒性。根据LD 50：ED 50比值，最安全的麻醉药是 依托咪酯和阿法沙龙。两者主要作为GABAA受体的有效和选择性调节剂， 主要抑制性神经递质门控离子通道。协同作用的麻醉剂组合- 通过降低脱靶毒性，对GABAA受体的cally也可以提供更大的安全性。远景目标 我们的研究目的是确定全身麻醉药在何处以及如何调节GABAA受体 活动我们之前对这个项目的研究已经帮助绘制了三组结构不同的transmits- 膜亚单位间结合位点分别为依托咪酯（咪唑）、阿法沙龙（神经类固醇）和 立体选择性催眠药巴比妥酸盐R-mTFD-MPAB，我们已经确定了这些位点的突变， 两者都模拟麻醉部位占据并阻碍麻醉剂结合。我们还发现依托咪酯， 异丙酚和戊巴比妥都作用于GABAA受体，包括突变体，与简单的两种状态雅阁。 变构共激动剂机制。然而，新的初步机制和突变体研究表明， 类似的模型不能解释阿法沙龙对GABAA受体的作用。此外，mTFD- 突变型受体上的MPAB或与野生型受体中的依托咪酯组合指示复杂的相互作用 不同麻醉部位之间的差异我们新的工作假设是 三种部位选择性麻醉剂依托咪酯、阿法沙龙和R-mTFD-MPAB明显影响功能性 受体状态，并且这些药物对在GABAA受体和 动物为了验证这些想法，我们建议使用分子模型，受体突变体，多个电生理， 逻辑方法，机制分析和一种新的动物模型来评估假设的关键方面。 在目标1中，我们将定义阿法沙龙和内源性神经类固醇THDOC和THDOC的机制 GABAA受体中的别孕烯醇酮不同于依托咪酯。在野生型和 将在卵母细胞（用于变构模型分析）和HEK 293细胞中进行突变GABAA受体的检测 (for快速动力学、状态依赖性和磷酸化研究）以解决多种可能性。在目标2中， 我们将量化和比较三种研究药物组合对野生型GABAA的影响， 受体，并评估和比较每组药物位点突变对所有三种药物作用的影响。 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“二进制” 变构位移分析。在目的3中，我们将确定组合药物对的药效学作用。 位点选择性麻醉剂在一种新的水生动物模型，斑马鱼幼虫。视频分析自发 活性和光运动反应将用于建立等效药物条件，作为 不同药物对的定量比较以及与目标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.