GENERAL ANESTHETIC SITE(S) OF ACTION ON GABA A RECEPTOR

γ氨基丁酸(GABA)是主要的抑制性神经递质 在脊椎动物的大脑中，这种神经递质的一个亚型 受体，GABAA受体是一个可能的药理靶点 全身麻醉剂。很明显，全麻药作用于GABAA 以高度特异的方式与受体结合。然而，地球表面的精确轨迹 GABAA受体多肽内的全身麻醉作用，或 “行动地点”，是未知的。 定义全身麻醉药的作用部位只能通过 全身麻醉药与麻醉药物相互作用的系统研究 表达的GABAA受体由已知亚单位组成，其中 氨基酸序列是已知的。GABAA受体仅由 在非洲爪哇卵母细胞中表达的Beta3或Rho1亚基表现出截然不同的 对GABAA和全身麻醉药的相反电生理反应， 因此，是分子解剖的极好的模型系统 行动地点。全身麻醉药能产生电流，而GABA则没有 对β3亚单位受体的影响。相反，Rho1亚基 受体对GABA有反应，但对全身麻醉药不起作用。的目标是 这项研究是为了检验全身麻醉药相互作用的假设 在与GABAA受体不同的部位以结构域特异的方式与其结合 GABA识别网站。这将通过以下方式实现 重组天然、突变或异构体的电生理实验 嵌合GABAA受体在非洲爪哇卵母细胞中的表达。表位标签 技术，结合免疫组织化学和Western印迹 分析，将被用来区分非功能和非表达 感受器。通过对氨基酸相似性和氨基酸的系统研究 不同亚基之间的差异，受体上的位置 一般直接开放经络所需的多肽 麻醉药，以及GABA诱导的增强所需的部位 海流，将被定义。接下来，这些生物的生理相关性 已确定的作用部位将在GABAA受体亚单位中进行测试 最有可能出现在大脑中的组合。 了解全麻药在哪里起作用对于未来是至关重要的 无副作用的全身麻醉药的开发 目前是导致临床发病率和死亡率的原因之一。此外， 一旦确定了行动地点。位点特异性逆转剂可能 可能会被开发出来。这样一种添加到药理上的 临床麻醉师可以使用的医疗设备是巨大的 意义，也许仅次于一般的发现 麻醉剂本身。

Gamma-amino-butyric-acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and one subtype of this neurotransmitter receptor, the GABAA receptor is a likely pharmacological target for general anesthetics. It is clear that general anesthetics act on the GABAA receptor in a highly specific manner. However the precise loci of the general anesthetic action within the GABAA receptor polypeptide, or the "sites-of-action", is unknown. Defining the sites-of-action of general anesthetics is only possible by a systematic study of the interactions between general anesthetics and expressed GABAA receptors composed of known subunits where the precise amino acid sequences are known. GABAA receptors consisting of only the beta3 or the rho1 subunits expressed in Xenopus oocytes show diametrically opposite electrophysiological responses to GABAA and general anesthetics, and therefore, are excellent model systems for the molecular dissection of the sites-of-action. General anesthetics induce currents, but GABA has no effect on the beta3 subunit receptors. Conversely, the rho1 subunit receptors respond to GABA, but not to general anesthetics. The goal of this study is to test the hypothesis that general anesthetics interact with the GABAA receptor in a domain specific manner at sites distinct from the GABA recognition site. This will be accomplished by electrophysiological experiments on recombinant native, mutated, or chimeric GABAA receptors expressed in Xenopus oocytes. The epitope-tag technique, in combination with immunohistochemistry and Western blot analysis, will be used to distinguish non-functional from non-expressed receptors. Through a systematic study of the amino acid similarities and differences between different subunits, the sites on the receptor polypeptide necessary for the direct opening of channels by general anesthetics, and the sites necessary for the potentiation of GABA induced currents, will be defined. Next, the physiological relevance of these identified sites-of-action will be tested in GABAA receptor subunit combinations most likely to be present in the brain. Understanding where general anesthetics work is essential for future development of general anesthetics without the side-effects which presently contribute to clinical morbidity and mortality. Furthermore, once the sites-of-action are defined. site specific reversal agents could possibly be developed. Such an addition to the pharmacological armamentarium available to clinical anesthesiologists is of immense significance, perhaps only secondary to the discovery of general anesthetics itself.