ANESTHETIC MECHANISMS AND GABA-A RECEPTOR STRUCTURE

GABA-A受体是主要的抑制性突触后 哺乳动物中枢神经系统中的受体。许多药物用于 全身麻醉诱导，包括苯二氮卓类、挥发性和 静脉内麻醉剂和巴比妥类药物增强GABA诱导的电流。的 增强的程度取决于特定的亚基-亚基组成， GABA-A受体在确定具有约束力的 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，由药物结合诱导的构象变化将需要高的分子量。 - 分辨率，X射线晶体结构。完整膜的结晶 蛋白质一直是一个难题， 整个GABA受体的结构将在不久的将来获得。一 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经产生， 将蛋白质的膜外结构域结晶为单独的， 水溶性蛋白质GABA受体的细胞外结构域含有 形成GABA和苯二氮卓结合位点的残基。主要目标 1）鉴定合适的表达系统， 过量产生GABA受体的细胞外结构域作为水溶性 蛋白质，2）表征蛋白质的物理和功能特性， 细胞外结构域蛋白和3）启动结晶试验， 蛋白我们已经截短了GABA rho-1亚基， 第一个跨膜结构域这些研究已经开始， rho-1亚基，因为它形成同源通道。259氨基酸 蛋白质是从果蝇S2细胞分泌的。它的流动性对蔗糖密度 梯度与其形成五聚体一致。一百毫克 可以从E. coli包涵体。 将研究再折叠胍增溶蛋白的条件。如果 成功的重折叠蛋白质将被表征并用于 结晶试验如果重折叠不成功， 真核表达系统，包括酵母和哺乳动物和昆虫细胞， 文化将受到考验。虽然该项目的风险很高，但其影响将 如果它导致细胞外的高分辨率结构， 离子型GABA受体亚单位的结构域。如果这种方法成功的话 这将为配体结合位点的结构提供新的认识 在细胞外结构域内。它还将提供深入了解 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.