General Anesthetic Sites in GABA-A Receptor Subunit Interfacial Pockets

GABA-A 受体亚基界面袋中的全身麻醉位点

基本信息

批准号：
9975188
负责人：
STUART A FORMAN
金额：
$ 34.06万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9975188
关键词：
Address Affect Agonist Allopregnanolone Alphaxolone Amino Acids Anesthetics Animal Behavior Animal Experiments Animal Model Animals Barbiturates Behavioral Binding Binding Sites Brain Cell Cycle Kinetics Cells Clinical Complex Coupling Cysteine Dangerousness Data Dependence Dose Drug Combinations Drug Modulation Drug toxicity Electrophysiology (science)Etomidate GABA Agonists GABA Modulators GABA-A Receptor GTP-Binding Protein alpha Subunits, Gs General Anesthesia General anesthetic drugs Goals Grant Imidazole Intravenous Investigation Ion Channel Gating Ions Kinetics Label Larva Lethal Dose 50 Link Maps Modeling Modification Molecular Structure Mutation Neurobiology Neurotransmitters Oocytes Patients Pentobarbital Pharmaceutical Preparations Pharmacodynamics Phosphorylation Propofol Research Research Project Grants Safety Site Standardization Structure Techniques Testing Toxic effect Training Zebrafish base drug development experimental study gamma-Aminobutyric Acid hypnotic improved interfacial molecular modeling mutant neurosteroids novel pharmacodynamic model receptor response synergism tetrahydrodeoxycorticosterone

项目摘要

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.

数以百万计的患者每年都会受到警惕训练有素的专家的全身麻醉管理一般麻醉药物毒性。基于LD50：ED50比的最安全麻醉药物是弹药和字母酮。两者都充当GABAA受体的潜在和选择性调节剂，大脑中主要的抑制性神经递质门控通道。作用协同作用的麻醉药的组合通过降低脱靶毒性，请致电GABAA接收器，也可以提供更大的安全性。长期目标我们的研究是定义一般麻醉的位置以及如何调节GABAA接收器活动。我们对该项目的先前研究帮助绘制了三个结构上不同的TransMem- Brane的亚基间结合位点，分别用于肌动蛋白（咪唑），字母酮（神经类固醇）和立体选择性催眠巴比妥酸R-MTFD-MPAB，我们已经确定了这些位点的突变都模仿麻醉部位的占用率并阻碍麻醉结合。我们还表明该突击素， proposalfol和五体性pentobarbital都按照一个简单的两态变构的联合机制。但是，新的初步机械和突变研究表明类似的模型不考虑GABAA受体上的字母作用。另外，MTFD-的影响 MPAB在突变受体上或与野生型受体中的蛋白酶结合表明复杂的相互作用在GABAA受体上的不同麻醉位点之间。我们的新工作假设是三种位点选择性麻醉药Emodate，Alphaxalone和R-MTFD-MPAB明显影响功能受体状态和这些药物的成对在GABAA受体和动物。为了测试这些想法，我们建议使用分子模型，接收器突变体，多个电物体 - 逻辑方法，机理分析和一种新的动物模型，以评估假设的关键方面。在AIM 1中，我们将定义字母和内源性神经类固醇的机制以及 GABAA受体中的异烷酮与依托氨基的不同之处。野生型和突变的GABAA受体将在两个卵母细胞（用于变构模型分析）和HEK293细胞中进行（用于快速动力学，状态依赖性和磷酸化研究）来解决多种可能性。在AIM 2中，我们将量化和比较在野生型Gabaa中组合三种研究药物对的影响受体和评估并比较每组药物部位突变对这三种作用的影响麻醉药。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” 变构转移分析。在AIM 3中，我们将建立组合对的药效学效应新型水生动物模型斑马鱼幼虫中的位点选择性麻醉药。赞助商的视频分析活性和光态反应将用于建立等效有效的药物条件作为基础不同药物对的定量比较以及与AIM 2的发现的相关性。