Cellular and Molecular Targets of General Anesthetics

全身麻醉药的细胞和分子靶点

• 批准号: 6678054
• 负责人: NEIL L. HARRISON
• 金额: $ 13.36万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6678054
• 关键词: Cellular; Molecular; Targets; General; Anesthetics

DESCRIPTION (provided by applicant): The long term goal of this project is to understand the mechanisms by which anesthetics modulate the operation of synaptic receptor channels. The central objective of the proposed research is to probe the mechanisms by which a widely-used anesthetic, propofol, activates and modulates a common inhibitory post-synaptic channel, the gamma-aminobutyric acid receptor (GABAAR). Although propofol has been administered to more than 300 million people world wide, its molecular mechanism of action on the GABAAR, its primary target, remains poorly understood. We propose to examine and quantify single-channel currents from recombinant GABAARs expressed in cell lines, using both cell-attached and outside-out patches. The effects of perturbations (e.g., mutations, ligands) will be analyzed in terms of changes in the fundamental gating allosteric constants. Specifically, we will determine the extents to which propofol alters the intrinsic gating equilibrium constant, the closed-channel equilibrium dissociation constant, and/or the open-channel equilibrium dissociation constant of the GABAAR. We also propose to probe the dynamics of GABAAR gating using linear free energy analysis. This approach should provide the sequence of events during the closed-open allostric transition, i.e., a map of which residues move early vs. late in the gating reaction. We will examine the interaction of propofol and propofol analogues at both the transmitter binding sites, and at a putative anesthetic binding site in the membrane domain of the protein. Further, we will explore the extents to which the pathway(s) of molecular motions that join the closed and open GABAAR conformations are similar in channels activated by ligand occupancy at either of these classes of binding site. These experiments should provide deep insight into the molecular mechanisms of action of an important anesthetic, propofol, and of an important synaptic receptor and target of anesthetic action, the GABAAR.

描述（由申请人提供）：本项目的长期目标是了解麻醉剂调节突触受体通道操作的机制。拟议研究的中心目标是探索广泛使用的麻醉剂丙泊酚激活和调节常见抑制性突触后通道γ-氨基丁酸受体（GABAAR）的机制。虽然丙泊酚已被全球超过3亿人使用，但其对GABAAR（其主要靶点）的分子作用机制仍知之甚少。我们建议检查和量化单通道电流从重组GABAARs在细胞系中表达，使用细胞附着和外向补丁。扰动的影响（例如，突变、配体）将根据基本门控变构常数的变化进行分析。具体而言，我们将确定丙泊酚改变GABAAR的内在门控平衡常数、闭合通道平衡解离常数和/或开放通道平衡解离常数的程度。我们还建议使用线性自由能分析来探测GABAAR门控的动力学。这种方法应该提供在关闭-打开别胃过渡期间的事件顺序，即，残基在门控反应中早期与晚期移动的图。我们将研究丙泊酚和丙泊酚类似物在递质结合位点和蛋白质膜结构域中假定的麻醉剂结合位点的相互作用。此外，我们将探讨在何种程度上的分子运动，加入封闭和开放的GABAAR构象的途径是相似的，在这些类别的结合位点的配体占用激活的通道。这些实验应该提供深入了解的分子机制的一个重要的麻醉剂，异丙酚，和一个重要的突触受体和麻醉剂的作用，GABAAR的目标。