NMR STUDIES OF MECHANISMS OF GENERAL ANESTHESIA

全麻药的分子作用机制仍然是个谜。配基门控离子通道的超家族被认为是全身麻醉药的主要靶点。我们自己和其他研究越来越清楚地表明，膜界面附近区域的两亲性是麻醉剂结合部位的统一属性(S)。因此，已有研究表明，全麻药而不是非固定剂(非麻醉剂)靶向于跨膜通道肽的两亲性界面残基，甘氨酸和γ-氨基丁酸A(GABAA)受体跨膜区II和III(TM2和TM3)的点突变可以完全消除甚至逆转这些受体对酒精和全身麻醉药的敏感性。对结构-功能关系的完整和详细的阐明将极大地促进我们对全身麻醉作用的理解，这在最近的过去甚至是无法想象的。这种竞争性更新将量化战略选择的全麻药和非固定剂对与人甘氨酸受体(GlyR)α1亚单位的TM2和TM3结构域的特定相互作用。最先进的蛋白质表达和纯化技术将与高分辨率和固态核磁共振(核磁共振)谱、圆二色谱(CD)和分子动力学模拟相结合，以实现三个特定目标：(1)表达GlyRα1亚基的野生型和突变的TM2和TM3片段，用于核磁共振结构研究。(2)在原子分辨率或接近原子分辨率下，确定人GlyRα1亚单位的功能性TM2和TM3片段以及相关的麻醉不敏感突变体的结构。(3)研究TM2和TM3中包含的全麻药结合的结构基序，并在所确定的结构框架内量化全麻药结合对通道动力学的影响，从而阐明控制通道对全麻药敏感性的结构要求。长期目标是将结构事件与全麻药引起的功能变化联系起来，为未来的活体和其他研究铺平道路，最终确定全麻药在中枢神经系统中的作用部位。

The molecular mechanisms of action of general anesthetics remain an enigma. A superfamily of ligand-gated ion channels has been implicated as the primary target sites for general anesthetics. It has become increasingly clear from our own and other studies that amphiphilicity in regions near the membrane interface is a unifying property of anesthetic binding site(s). Thus, general anesthetics, but not nonimmobilizers (nonanesthetics), have been shown to target amphiphilic interfacial residues of transmembrane channel peptides, and point mutations in the transmembrane domains II and III (TM2 and TM3) of glycine and gamma- aminobutyric acidA (GABAA) receptors can completely abolish or even reverse the sensitivity of these receptors to alcohol and general anesthetics. Complete and detailed elucidation of the structure-function relationship will dramatically advance our understanding of general anesthetic action beyond what was even imaginable in the recent past. This competitive renewal will quantify specific interactions of strategically selected pairs of general anesthetics and nonimmobilizers with the TM2 and TM3 domains of the alpha1 subunit of human glycine receptors (GlyR). State-of-the-art protein expression and purification techniques will be coupled with high-resolution and solid-state nuclear magnetic resonance (NMR) spectroscopy, circular dichroism (CD), and molecular dynamic simulations to accomplish three specific aims: (1) To express the wild-type and mutated TM2 and TM3 segments of GlyR alpha1 subunit for structural study by NMR. (2) To determine, at or near atomic resolution, the structures of the functional TM2 and TM3 segments of the human GlyR alpha1 subunit and the associated anesthetic-insensitive mutants. (3) To investigate the structural motifs contained in TM2 and TM3 for general anesthetic binding, and to quantify the effects of general anesthetic binding on channel dynamics within the determined structural frame, thereby elucidating the structural requirement that controls the channel sensitivity to general anesthetics. The long-term goal is to relate the structural events to functional changes caused by general anesthetics, paving the way for future in vivo and other studies to finally identify the sites of action of general anesthetics in the central nervous system.