PHOTOLABELING OF ANESTHETIC STEROID BINDING SITES

麻醉类固醇结合位点的照片标记

• 批准号: 6501516
• 负责人: ALEX S. EVERS
• 金额: $ 8.62万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6501516
• 关键词: GABA receptor; anesthetics; animal tissue; anticonvulsants; binding sites; brain; chemical structure; convulsants; electrophoresis; protein sequence; sedative /hypnotic; steroid hormone analog; tissue /cell culture; western blottings

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.

某些内源性类固醇及其合成类似物（神经活性 类固醇）对中枢神经产生深远而快速的影响 系统范围从全身麻醉到癫痫发作。 虽然这些 效果被认为是类固醇与特定物质相互作用的结果 GABAA 受体的结合位点，方法尚未确定 可用于识别和表征这些假定的神经活性类固醇 结合位点。 该提案是基于发展，由 申请人实验室的光亲和标记试剂（PAL-1） 推定的神经活性类固醇结合位点。 PAL-1已被证明可以 调节 GABA 激活的氯电流浓度低于 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试剂。 本项目将使用 PAL-1 实现三个具体目标：（10 蛋白质和蛋白质 具有神经活性类固醇特异性结合位点的亚基将是 确定，并且类固醇结合的化学计量和亲和力将是 测量。 为了实现这一目标，PAL-1 的放射性类似物 将进行合成并进行光标记实验 对纯化的 GABAA 受体和脑组织的影响；标记蛋白质 通过蛋白质电泳、蛋白质测定来鉴定 放射性和免疫印迹的结合。 (2) 确定 神经活性类固醇结合位点与位点之间的关系 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、惊厥剂和 脑组织中 PAL-1 标记的抗惊厥药。 (3) 确定 PAL-1 标记的氨基酸序列，光标记研究将 也可以对转染特定的培养细胞进行 GABAA 受体亚基的组合。 标记的肽将是 通过免疫沉淀和微测序纯化。 这 从该项目获得的信息将提供背景 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的中枢神经系统功能 和疾病和； 2）开发新的药剂，包括 强效类固醇麻醉剂，副作用最小，新颖 抗惊厥药和神经活性类固醇拮抗剂。