ANESTHETIC MECHANISMS AND GABA-A RECEPTOR STRUCTURE

麻醉机制和 GABA-A 受体结构

• 批准号: 6387316
• 负责人: Myles H. Akabas
• 金额: $ 12.56万
• 依托单位: ALBERT EINSTEIN COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6387316
• 关键词: GABA receptor; anesthetics; binding sites; bioreactors; conformation; crystallization; drug receptors; microorganism culture; pharmacokinetics; protein folding; protein structure function; tissue /cell culture; transfection; water solubility

The GABA-A receptors are the major inhibitory post-synaptic receptors in the mammalian central nervous system. Many drugs used in the induction of general anesthesia, including benzodiazepines, volatile and intravenous anesthetics and barbiturates potentiate GABA-induced currents. The extent of potentiation depends on the specific subunit-subtype composition of the GABA-A receptors. Progress has been made towards identifying the binding sites for these drugs. Mutagenesis and photoaffinity labeling studies identified some amino acids that may form the binding sites for these drugs. Understanding the three dimensional structure of the drug binding sites and the conformational changes induced by drug binding, however, will require a high -resolution, x-ray crystal structure. The crystallization of integral membrane proteins has been a difficult problem and it is unlikely that a crystal structure of the entire GABA receptor will be obtained in the near future. A partial solution to obtaining high-resolution, three-dimensional structural information about integral membrane proteins has been to produce and crystallize the extra-membrane domains of the proteins as separate, water-soluble proteins. The extracellular domain of the GABA receptors contains residues that form the GABA and benzodiazepine binding sites. The major goals of this application are 1) to identify a suitable expression system that will overproduce the extracellular domain of the GABA receptor as a water-soluble protein, 2) to characterize the physical and functional properties of the extracellular domain protein and 3) to initiate crystallization trials with the protein. We have truncated the GABA rho-1 subunit just prior to the start of the first membrane-spanning domain. These studies have been initiated with the rho-l subunit because it forms homomeric channels. The resultant 259 amino acid protein is secreted from Drosophila S2 cells. Its mobility on a sucrose-density gradient is consistent with its forming a pentamer. One hundred milligram quantities can be produced and purified from E. coli inclusion bodies. Conditions to refold the guanidine-solubilized protein will be investigated. If successful the refolded protein will be characterized and used in crystallization trials. If refolding is unsuccessful over-expression in eukaryotic expression systems including yeast and mammalian and insect cells in culture will be tried. While the risk of this project is high the impact would also be high if it results in a high-resolution structure of the extracellular domain of an ionotropic GABA receptor subunit. If this approach is successful it will provide new understanding of the structure of the ligand binding sites within the extracellular domain. It will also provide insights into the conformational changes induced by agonist and drug binding and thereby provide an understanding of anesthetic action at a molecular level.

GABA-A 受体是主要的突触后抑制性受体 哺乳动物中枢神经系统中的受体。许多药物都用于 全身麻醉的诱导，包括苯二氮卓类药物、挥发性和 静脉麻醉药和巴比妥类药物会增强 GABA 诱导的电流。这 增强的程度取决于具体的亚基-亚型组成 GABA-A 受体。在确定约束力方面已取得进展 这些药物的网站。诱变和光亲和标记研究 确定了一些可能形成这些药物结合位点的氨基酸。 了解药物结合位点的三维结构和 然而，药物结合引起的构象变化需要高 -分辨率，X射线晶体结构。积分膜结晶 蛋白质一直是一个难题，晶体不太可能 整个GABA受体的结构将在不久的将来获得。一个 获得高分辨率三维结构的部分解决方案 关于整合膜蛋白的信息已被生产和 将蛋白质的膜外结构域结晶为单独的， 水溶性蛋白质。 GABA 受体的胞外结构域包含 形成 GABA 和苯二氮卓结合位点的残基。主要目标 该应用的目的是 1) 确定合适的表达系统 过量产生水溶性 GABA 受体的胞外结构域 蛋白质，2) 表征其物理和功能特性 胞外结构域蛋白和 3) 启动结晶试验 蛋白质。我们在开始之前截断了 GABA rho-1 亚基 第一个跨膜结构域。这些研究是由 rho-l 亚基，因为它形成同聚通道。所得259个氨基酸 蛋白质由果蝇 S2 细胞分泌。其在蔗糖密度下的迁移率 梯度与其形成五聚体一致。一百毫克 可以从大肠杆菌包涵体中生产和纯化大量的。 将研究重新折叠胍溶解的蛋白质的条件。如果 成功后，重折叠蛋白将被表征并用于 结晶试验。如果重折叠不成功则过度表达 真核表达系统，包括酵母和哺乳动物和昆虫细胞 文化将被尝试。虽然该项目风险较高，但影响 如果它导致细胞外结构的高分辨率，那么它也会很高 离子型 GABA 受体亚基的结构域。如果这个方法成功的话 它将提供对配体结合位点结构的新理解 在细胞外域内。它还将提供有关 由激动剂和药物结合诱导的构象变化，从而提供 在分子水平上了解麻醉作用。