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类固醇与GABA-A受体上特定结合位点的相互作用引起的，但分子生物学研究未能确定可能形成结合位点的候选区域或残基。该项目的总体目标是确定和表征与神经活性类固醇相互作用产生抑制（麻醉）作用的结合位点。两种新型神经类固醇类似物光亲和标记试剂CW12 （sa -还原类固醇）和CW14 （5p-还原类固醇）已经开发出来以实现这一目标。CW12和CW14都是GABA-A受体功能的有效调节剂。当这些试剂暴露在350 nM的紫外光下时，它们形成一个活性基团（一个碳烯），使它们能够共价地附着在（光标记）它们的结合位点上。脑膜中GABA-A受体功能的测定提供了强有力的证据，证明CW12和CW14有效和选择性地光标记了负责调节GABA-A受体功能的神经活性类固醇结合位点。这个项目有两个具体目标。首先是鉴定和表征大鼠脑中由CW12和/或CW14光标记的GABA-A受体相关蛋白。第二个目的是通过光标记转染了表位标记GABA-A受体的各种组合的HEK细胞，并将光标记的功能效应与被光标记的特定亚基相关联，来确定GABA-A受体亚基对神经活性类固醇结合和作用的特异性。在这两个目标中，光标记蛋白将使用免疫沉淀和双向电泳分离，然后使用串联质谱鉴定和测序。从这个项目中获得的信息将提供背景知识和工具：(1)确定内源性神经类固醇如何调节健康和疾病中的中枢神经系统功能；(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、抗焦虑药和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.