PHOTOLABELING OF ANESTHETIC STEROID BINDING SITES

计划2 某些内源性类固醇和它们的合成类似物（神经活性类固醇）产生深刻的， 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 被认为是由类固醇与GABA-A受体上的特异性结合位点相互作用引起的。的 本项目的总体目标是鉴定和表征GABA-A受体的结合位点， 其中神经活性类固醇相互作用以产生它们的抑制（麻醉）作用。两种新型 神经类固醇类似物光亲和标记试剂，CW 12（Sa-还原类固醇）和CW 14（5 β- 减少的类固醇）已被开发以实现该目标。CW 12和CW 14都是有效的， GABA-A受体功能的有效调节剂。当这些试剂暴露于UV光时， 350 nM，它们形成一个反应基团（卡宾），使它们共价连接到（光标记） 它们的结合位点。CW 12光标记大鼠脑和培养细胞中的GABA-A受体亚基。 GABA-A受体功能的测定表明，CW 12和CW 14选择性地光标记GABA-A受体。 负责调节GABA-A受体功能的神经活性类固醇结合位点。的目的 本项目旨在鉴定被CW 12和/或CW 14光标记的GABA-A受体亚单位 并鉴定被光标记试剂修饰的特定氨基酸残基。 光标记研究将在用各种转染的培养的哺乳动物细胞中进行。 表位标记的GABA-A受体的组合和大鼠脑组织。光标记GABA-A 将使用免疫沉淀分离受体，并用蛋白酶消化以产生类固醇， 修饰的和未修饰的肽。然后使用毛细管电泳法分析类固醇修饰的肽。 高效液相色谱-线性四极离子阱-傅立叶变换离子回旋加速器 质谱法测定它们的序列并鉴定修饰的氨基酸。的 从本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 包括具有最小副作用的强效甾体麻醉剂的新型药物 抗惊厥药和抗焦虑药以及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.