PHOTOLABELING OF ANESTHETIC STEROID BINDING SITES

项目2 某些内源性类固醇及其合成类似物(神经活性类固醇)能产生深刻和 对中枢神经系统的快速影响，从全身麻醉到癫痫发作。这些影响 被认为是类固醇与GABA-A受体上特定结合部位相互作用的结果。这个 这个项目的总体目标是识别和表征GABA-A受体上的结合位点 哪些神经活性类固醇相互作用而产生抑制(麻醉)作用。两部小说 神经类固醇类光亲和标记试剂CW12(Sa-还原类固醇)和CW14(5p- (减少类固醇)已被开发出来以实现这一目标。CW12和CW14都是强效的 GABA-A受体功能的有效调节剂。当这些试剂暴露在紫外光下时 350 nm，它们形成一个活性基团(卡宾)，使它们能够共价连接到(光标记物)上 它们的结合部位。CW12光标记大鼠脑和培养细胞中的GABA-A受体亚单位。 GABA-A受体功能测定表明，CW12和CW14选择性地标记了 神经活性类固醇结合位点(S)，负责调节GABA-A受体功能。的目的是 本项目旨在鉴定由CW12和/或CW14光标记的GABA-A受体亚基 并鉴定光标记试剂修饰的特定氨基酸残基。 将在培养的哺乳动物细胞中进行光标记研究，这些细胞被转染了不同的 表位标记的GABA-A受体和大鼠脑组织中的组合。光标记GABA-A 受体将用免疫沉淀法分离，并用蛋白酶消化产生类固醇- 修饰和未修饰的多肽。然后用毛细管对类固醇修饰的多肽进行分析。 高效液相色谱-线性四极离子陷阱-傅里叶变换离子回旋加速器 用质谱仪测定它们的序列并鉴定修饰后的氨基酸。这个 从该项目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在健康和疾病中调节中枢神经系统功能；(2)开发新的 包括副作用最小的有效类固醇麻醉剂的药物，新 抗惊厥药、抗焦虑药和神经活性类固醇拮抗剂。

PROJECT 2 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. These effects are thought to result from steroid interactions with specific binding sites on the GABA-A receptor. The overall goal of this project is to identify and characterize the binding sites on the GABA-A receptor 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. CW12 photolabels GABA-A receptor subunits both in rat brain and in cultured cells. Assays of GABA-A receptor function demonstrate that CW12 and CW14 selectively photolabel the neuroactive steroid binding site(s) responsible for modulation of GABA-A receptor function. The aim of this project is to identify the GABA-A receptor subunits that are photolabeled by CW12and/or CW14 and to identify the specific amino acid residues that are modified by the photolabeling reagents. Photolabeling studies will be performed in cultured mammalian cells transfected with various combinations of epitope-tagged GABA-A receptors and in rat brain tissue. Photolabeled GABA-A receptors will be isolated using immunoprecipitation and digested with proteases to generate steroid- modified and unmodified peptides. The steroid-modified peptides will then be analyzed using capillary high-performance liquid chromatography-linear quadrupole ion trap-Fourier transform ion cyclotron mass spectrometry to determine their sequence and to identify the modified amino acids. 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.