Project 3: Anesthetic Mechanisms on GABAA Receptors

项目3：GABAA受体的麻醉机制

• 批准号: 7777111
• 负责人: STUART A FORMAN
• 金额: $ 28.44万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2014-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7777111
• 关键词: Accounting; Affect; Affinity; Agonist; Amino Acids; Anesthetics; Barbiturates; Binding; Biological Assay; Chemicals; Chemosensitization; Clinical; Cysteine; DNA Sequence Rearrangement; Data; Development; Electrophysiology (science); Equilibrium; Etomidate; Gated Ion Channel; General anesthetic drugs; Homologous Gene; Homology Modeling; Hydrophobicity; Kinetics; Ligands; Link; Measures; Membrane; Modeling; Modification; Molecular; Mutagenesis; Mutation; Neurons; Operative Surgical Procedures; Pentobarbital; Pharmaceutical Preparations; Probability; Procedures; Propofol; Role; Side; Simulate; Site; Specificity; Synapses; Testing; Therapeutic; Time; Work; analog; barbituric acid salt; base; desensitization; gamma-Aminobutyric Acid; mutant; novel; programs; receptor; research study; spatial relationship; therapeutic target

General anesthetics are widely used during surgery and other clinical procedures, yet they are extremely dangerous and their mechanisms remain poorly understood. Understanding their actions at the molecular level will enable development of new drugs with more therapeutic specificity. The overall aim of this project is to define the mechanisms of potent general anesthetics at known therapeutic targets: synaptic and extrasynaptic GABAA receptors. Photolabeling, structural homology modeling, mutant studies, and mechanistic analysis have established structural and functional models for etomidate actions at synaptic aiP2Y2L receptors, providing a new paradigm for other potent anesthetics such as propofol and barbiturates, and extra-synaptic GABAARS containing different subunits. Our working hypothesis is that at synaptic cci(32/372 and extrasynaptic OAPSS GABAA receptors, different potent anesthetics bind to distinct sub-regions of intra-membrane pockets formed at a-p subunit interfaces, with each subunit contributing a channel-lining transmembrane M2 domain and one other: a-M1 and P-M3. Anesthetic binding facilitates rearrangement of these pockets, stabilizing open channel states, thereby reducing neuronal activity. We propose to electrophysiologically study expressed GABAARS of defined subunit composition, developing a detailed mechanism of anesthetic actions at the macrocurrent level and determining how structural changes in the putative drug pockets affect anesthetic interactions and channel gating. Specific Aim 1 is to investigate anesthetic-photolabeled amino acids in aip2/3y2L (synaptic) GABAARS and their roles in channel gating and the affinity/efficacy of etomidate, propofol, and pentobarbital. Mutagenesis, including cysteine mutagenesis and state-dependent modification, together with electrophysiological analysis based on an equilibrium co¿ agonist gating model will be used. Specific Aim 2 is to build an allosteric-kinetic model for etomidate modulation of aiP2/3'y2L GABAARS and to develop allosteric co-agonist models for propofol and pentobarbital. Macrocurrent kinetic electrophysiology will be performed using an "artificial synapse" for rapid application of GABA and anesthetics, and kinetic models will be assessed for their ability to quantitatively account for the data. Specific Aim 3 is to extend the above approaches to a4p35 (extrasynaptic) GABAARS. Specific Aim 4 is to collaborate with other PPG projects by assessing new photolabel anesthetics and guiding time-resolved photolabeling experiments. RELEVANCE (See instmctions):

全身麻醉剂在手术和其他临床程序期间被广泛使用，但它们极其昂贵。 危险，其机制仍然知之甚少。了解它们在分子水平上的行为 水平将使开发具有更高治疗特异性的新药成为可能。该项目的总体目标是 目的是确定强效全身麻醉剂在已知治疗靶点的作用机制：突触和 突触外GABAA受体。光标记、结构同源性建模、突变体研究和 机制分析已经建立了依托咪酯作用于突触的结构和功能模型， aiP 2 Y2 L受体，为其他强效麻醉剂如丙泊酚和巴比妥酸盐提供了新的范例， 和含有不同亚基的突触外GABAARS。我们的工作假设是，在突触 cci（32/372）和突触外OAPSS GABAA受体，不同的强效麻醉剂结合到不同的亚区， 在a-p亚基界面处形成的膜内口袋，每个亚基贡献通道衬里 跨膜M2结构域和另一个：α-M1和P-M3。麻醉剂的结合有助于 这些口袋，稳定开放通道状态，从而减少神经元活动。我们建议 电生理学研究表达了GABAARS的定义亚基组成，开发了一个详细的 在宏电流水平的麻醉作用机制，并确定如何在结构变化， 推定的药物袋影响麻醉剂相互作用和通道门控。具体目标1是调查 aip 2/3 y2 L（突触）GABAARS中的麻醉光标记氨基酸及其在通道门控和 依托咪酯、丙泊酚和戊巴比妥的亲和力/功效。诱变，包括半胱氨酸诱变 和状态依赖的修改，以及基于平衡CO的电生理分析。 将使用激动剂门控模型。具体目标二是建立依托咪酯的变构动力学模型 调节α 1 β 2/3 γ 2L GABAARS，并开发丙泊酚和戊巴比妥的变构共激动剂模型。 将使用“人工突触”进行宏电流动力学电生理学，以快速应用 将评估GABA和麻醉剂以及动力学模型定量说明 数据具体目标3是将上述方法扩展到a4 p35（突触外）GABAARS。具体目标4 是通过评估新的光标记麻醉剂和指导时间分辨， 光标记实验。 相关性（见说明）：