Cellular and Molecular Targets of General Anesthetics

描述（由申请人提供）：该项目的长期目标是了解麻醉药调节突触受体通道运作的机制。本研究的主要目的是探讨一种广泛使用的麻醉剂异丙酚激活和调节一种常见的抑制性突触后通道——γ -氨基丁酸受体（GABAAR）的机制。尽管异丙酚已在全球范围内应用于3亿多人，但其对GABAAR（其主要靶点）的分子作用机制仍知之甚少。我们建议使用细胞贴附和外接贴片来检测和量化细胞系中表达的重组GABAARs的单通道电流。扰动（例如，突变，配体）的影响将根据基本门控变构常数的变化进行分析。具体来说，我们将确定异丙酚改变GABAAR的内在门控制平衡常数、封闭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索GABAAR门控的动力学。这种方法应该提供闭开异位过渡期间的事件序列，即，在门控反应中哪些残基移动早与晚的地图。我们将研究异丙酚和异丙酚类似物在递质结合位点和在蛋白质膜结构域中假定的麻醉结合位点的相互作用。此外，我们将探索在这两类结合位点的配体占用激活的通道中，连接封闭和开放GABAAR构象的分子运动途径在何种程度上相似。这些实验将为深入了解重要的麻醉剂异丙酚和重要的突触受体和麻醉作用靶点GABAAR的分子机制提供帮助。

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.