PHYSIOLOGICAL STUDIES OF NEUROSTEROID ANALOGUES

项目4 神经活性类固醇是γ-氨基丁酸-A受体（GABAR）的有效调节剂， 在低浓度下增强GABA的作用并直接激活GABAR氯离子通道， 在较高浓度下缺乏GABA。其他类固醇，特别是带有带电取代基的类固醇 在CS-位，是GABAR的负调节剂和N-甲基-D- 天冬氨酸受体（NMDAR）。目前的证据表明，GABAR增强和NMDAR抑制 代表了临床麻醉中的两种关键机制。在过去的几年里，我们开发了一些工具， 帮助我们探索神经活性类固醇对GABAR的影响，包括一系列新型类固醇 对映体和荧光类固醇，提供了重要的新信息，细胞内和 这些物质的膜内积累。其中一种荧光类固醇只能增强GABAR 当暴露于可见光（~ 480 nm）时，提供了开发一类新的GABA能 可以在细胞或大脑的特定区域激活的调节剂。在目前的建议中，我们将扩大 我们对神经活性类固醇的研究旨在实现四个目标：1。检查细胞膜和受体 有助于GABA增强3-α-羟基类固醇的作用的机制; 2.审查 膜机制有助于C3-硫酸化类固醇和3-β-羟基类固醇的作用， GABAR; 3.研究光活性类固醇对GABAR的影响机制; 4. 获得有关GABAR和NMDAR上类固醇结构要求的新信息。这些 研究将使用生理学和细胞成像方法相结合，以检查类固醇的影响， 培养的大鼠海马神经元和HEK细胞以及表达确定的GABAR的非洲爪蟾卵母细胞 亚基，并将涉及结构活性和机制研究的结合，根据我们的初步研究， 观察与新的荧光和光敏类固醇类似物。这些研究有可能 提供有关CMS中神经活性类固醇作用的新信息，并更好地了解 类固醇介导的麻醉机制。

PROJECT 4 Neuroactive steroids are effective modulators of gamma-aminobutyric acid-A receptors (GABARs), augmenting the actions of GABA at low concentrations and directly activating GABAR chloride channels in the absence of GABA at higher concentrations. Other steroids, particularly those with charged substituents at the CS-position, are negative modulators of GABARs and either potentiators or inhibitors of N-methyl-D- aspartate receptors (NMDARs). Present evidence indicates that GABAR potentiation and NMDAR inhibition represent two key mechanisms in clinical anesthesia. Over the several years, we have developed tools that have helped us to probe the effects of neuroactive steroids on GABARs, including a series of novel steroid enantiomers and fluorescent steroids that have provided important new information about intracellular and intramembranous accumulation of these agents. One of the fluorescent steroids only potentiates GABARs when exposed to visible (-480 nm) light, offering the potential to develop a new class of GABAergic modulators that can be activated in specific regions of cells or brain. In the present proposal, we will extend our studies of neuroactive steroids by addressing four goals: 1. To examine membrane and receptor mechanisms contributing to the effects of GABA potentiating 3-alpha-hydroxysteroids; 2. To examine membrane mechanisms contributing to the effects of C3-sulfated steroids and 3-beta-hydroxysteroids on GABARs; 3. To examine mechanisms contributing to the effects of photoactive steroids on GABARs; and 4. To gain new information about structural requirements for steroids on GABARs and NMDARs. These studies will use a combination of physiological and cellular imaging methods to examine steroid effects in cultured rat hippocampal neurons, and HEK cells and Xenopus oocytes expressing defined GABAR subunits, and will involve a combination of structure-activity and mechanistic studies based upon our initial observations with the novel fluorescent and photoactive steroid analogues. These studies have the potential to provide new information about neuroactive steroid effects in the CMS and a better understanding of mechanisms involved in steroid-mediated anesthesia.