Anesthetics Cellular and Molecular Actions

描述（由申请人提供）：某些内源性类固醇及其合成类似物（神经活性类固醇）对中枢神经系统产生深刻而迅速的影响，从全身麻醉到癫痫发作。虽然这些作用被认为是由于类固醇与GABA-A受体上的特异性结合位点的相互作用，但分子生物学研究未能确定可能有助于结合位点的候选区域或残基。该项目的总体目标是识别和表征与神经活性类固醇相互作用以产生其抑制（麻醉）作用的结合位点。为了实现这一目标，开发了两种新型神经甾体类似物光亲和标记试剂CW 12（Sa还原的甾体）和CW 14（5 p还原的甾体）。CW 12和CW 14都是GABA-A受体功能的强效和有效的调节剂。当这些试剂暴露于350 nM的紫外光时，它们形成一个反应基团（卡宾），使它们能够共价连接（光标记）它们的结合位点。 对脑膜中GABA-A受体功能的测定提供了强有力的证据，即CW 12和CW 14有效地和选择性地光标记负责调节GABA-A受体功能的神经活性类固醇结合位点。该项目有两个具体目标。第一个是确定和表征的GABA-A受体相关的蛋白，CW 12和/或CW 14的光标记在大鼠脑。第二个目的是确定GABA-A受体亚基特异性的神经活性类固醇的结合和行动，通过光标记HEK细胞转染的各种组合的表位标记的GABA-A受体和相关的功能效应的光标记的特定亚基的光标记。在这两个目标中，将使用免疫沉淀和二维电泳分离光标记的蛋白质，然后使用串联质谱法进行鉴定和测序。 从该项目中获得的信息将提供背景知识和工具，以：（1）确定内源性神经类固醇如何调节健康和疾病中的CNS功能;（2）开发新的药物制剂，包括副作用最小的强效类固醇麻醉剂，新型抗惊厥药和抗焦虑药以及神经活性类固醇拮抗剂。

DESCRIPTION (provided by applicant): Certain endogenous steroids and their synthetic analogues (neuroactive steroids) produce profound and rapid effects on the central nervous system ranging from general anesthesia to seizures. While these effects are thought to result from steroid interactions with specific binding sites on the GABA-A receptor, molecular biological studies have failed to identify candidate regions or residues that might contribute to a binding site. The overall goal of this project is to identify and characterize the binding sites with which neuroactive steroids interact to produce their inhibitory (anesthetic) effects. Two novel neurosteroid analogue photoaffinity labeling reagents, CW12 (a Sa-reduced steroid) and CW14 (a 5p-reduced steroid) have been developed to achieve this goal. Both CW12 and CW14 are potent and efficacious modulators of GABA-A receptor function. When these reagents are exposed to UV light at 350 nM, they form a reactive group (a carbene) that allows them to covalently attach to (photolabel) their binding site. Assays of GABA-A receptor function in brain membranes provide strong evidence that CW12 and CW14 efficiently and selectively photolabel the neuroactive steroid binding site(s) responsible for modulation of GABA-A receptor function. This project has two specific aims. The first is to identify and characterize the GABA-A receptor-associated proteins that are photolabeled by CW12 and/or CW14 in rat brain. The second aim is to determine the GABA-A receptor subunit specificity of neuroactive steroid binding and action by photolabeling HEK cells transfected with various combinations of epitope-tagged GABA-A receptors and correlating the functional effects of photolabeling with the specific subunits that are photolabeled. In both aims, photolabeled proteins will be isolated using immunoprecipitation and two-dimensional electrophoresis and then identified and sequenced using tandem mass spectrometry. The information gained from this project will provide the background knowledge and tools to: (1) determine how endogenous neurosteroids modulate CNS function in health and disease and; (2) develop new pharmaceutical agents including potent steroidal anesthetics with minimal side effects, novel anticonvulsants and anxiolytics and neuroactive steroid antagonists.