ANESTHETIC MECHANISMS AND GABA-A RECEPTOR STRUCTURE

GABA-A受体是突触后的主要抑制性物质 哺乳动物中枢神经系统中的受体。许多药物被用在 全麻诱导，包括苯二氮类药物，挥发性和 静脉麻醉剂和巴比妥酸盐可增强GABA诱导的电流。这个 增强的程度取决于特定的亚基-亚型组成 GABA-A受体。在确定绑定方面取得了进展 这些毒品的网站。诱变和光亲和标记研究 确定了可能形成这些药物结合部位的一些氨基酸。 了解药物结合部位的三维结构和 然而，药物结合引起的构象变化将需要很高的 -分辨率，x射线晶体结构。整体膜的结晶 蛋白质一直是个难题，而且晶体不太可能 整个GABA受体的结构将在不久的将来得到。一个 获取高分辨率三维结构的部分解 关于完整的膜蛋白的信息已经被生产和 将蛋白质的膜外结构域结晶为独立的， 水溶性蛋白质。GABA受体的胞外区包含 形成GABA和苯二氮卓结合部位的残基。主要目标 本申请的目的是：1)确定合适的表达系统，该系统将 过量生产GABA受体的胞外区作为一种水溶性 蛋白质，2)表征蛋白质的物理和功能特性 胞外结构域蛋白和3)启动结晶试验 蛋白。我们已经截断了GABA Rho-1亚基，就在 第一个跨膜结构域。这些研究是与 Rho-L亚基，因为它形成同质通道。生成的259个氨基酸 果蝇S2细胞分泌蛋白质。它在蔗糖密度上的流动性 梯度与其形成的五聚体相一致。100毫克 可以从大肠杆菌包涵体中大量生产和纯化。 将研究重折叠胍增溶蛋白的条件。如果 成功的复性蛋白将被表征并用于 结晶试验。如果重折叠不成功，则在 酵母、哺乳动物和昆虫细胞的真核表达系统 文化将会受到考验。虽然这个项目的风险很高，但其影响将 如果它产生高分辨率的细胞外结构，也是高的 离子型GABA受体亚单位的结构域。如果这种方法成功了 这将对配体结合部位的结构提供新的理解 在胞外区域内。它还将提供对 激动剂和药物结合引起的构象变化，从而提供 从分子水平理解麻醉剂的作用。

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.