PHOTOLABELING OF ANESTHETIC STEROID BINDING SITES

某些内源性类固醇及其合成类似物(神经活性 类固醇)对中枢神经产生深刻而迅速的影响 从全身麻醉到癫痫发作的各种系统。而这些 效应被认为是由于类固醇与特定的 GABAA受体的结合部位，方法还没有 可用于鉴定和表征这些假定的神经活性类固醇 结合部位。这项建议是基于由 申请人实验室，光亲和标记剂(PAL-1)，用于 可能的神经活性类固醇结合位点(S)。PAL-1已被证明 以低于以下浓度调制GABA激活的氯电流 5纳摩尔，并具有适当的光化学性质 作为一种有效的光标记剂。该项目将使用 PAL-1可实现三个特定目标：(10蛋白质和蛋白质 与神经活性类固醇有特定结合位点的亚基将是 类固醇结合的化学计量和亲和力将是 量过了。为了实现这一目标，一种PAL-1的放射性类似物 将被合成，并将进行光标记实验 在纯化的GABAA受体和脑组织上；标记蛋白 将通过蛋白质电泳法、蛋白质测量法进行鉴定 放射性掺入和免疫印迹。(二)确定 神经活性类固醇结合部位与部位的关系 对于其他神经活性药物的作用，将进行研究 检查各种麻醉剂、镇静剂、惊厥药和 抗惊厥药物对脑组织PAL-1标记的影响。(三)确定 PAL-1标记的氨基酸序列，光标记研究将 也可以在培养的细胞上进行转导特定基因的实验。 GABAA受体亚基的组合。标记的多肽将是 用免疫沉淀法纯化，并进行微序列测定。The the the 从这个项目中获得的信息将提供背景信息 知识和药理学工具：1)解决以下问题 内源性神经活性类固醇如何调节健康人的中枢神经系统功能 和疾病；2)开发新的药物制剂，包括 副作用最小的有效类固醇麻醉药，新 抗惊厥药和神经活性类固醇拮抗剂。

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 of the GABAA receptor, methods have not been available to identify and characterize these putative neuroactive steroid binding sites. This proposal is based on the development, by the applicant lab, of a photoaffinity labeling reagent (PAL-1) for the putative neuroactive steroid binding site(s). PAL-1 has been shown to modulate GABA-activated chloride currents at concentrations less than 5 nanomolar, and to have appropriate photochemical properties to function as an efficient photolabeling reagent. This project will use PAL-1 to achieve three specific goals: (10 The proteins and proteins subunits that have specific binding sites for neuroactive steroids will be identified, and the stoichiometry and affinity of steroid binding will be measured. To accomplish this goal, a radioactive analogue of PAL-1 will be synthesized and photolabeling experiments will be performed on purified GABAA receptors and on brain tissue; labeled proteins will be identified by protein electrophoresis, measurement of protein incorporation of radioactivity and immunoblotting. (2) To determine the relationship between neuroactive steroid binding sites and the sites of action of other neuroactive drugs, studies will be performed examining the effects of various anesthetics, sedatives, convulsants and anti-convulsants on PAL-1 labeling in brain tissue. (3) To determine the amino acid sequences labeled by PAL-1, photolabeling studies will also be performed on cultured cells transfected with specific combinations of GABAA receptor subunits. Labeled peptides will be purified by immunoprecipitation and microsequenced. The information gained from this project will provide the background knowledge and pharmacologic tools to: 1) approach the question of how endogenous neuroactive steroids modulate CNS function in health and disease and; 2) to develop new pharmaceutical agents including potent steroidal anesthetics with minimal side effects, novel anticonvulsants and neuroactive steroid antagonists.