PHYSIOLOGICAL STUDIES OF NEUROSTEROID ANALOGUES

项目4 神经活性类固醇是γ-氨基丁酸-A受体(GABA)的有效调节剂， 增强低浓度GABA的作用及直接激活GABA氯离子通道 较高浓度的GABA的缺失。其他类固醇，特别是那些带电荷取代基的类固醇 在CS-位是GABA的负调节剂和N-甲基-D-D的增效剂或抑制剂。 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个关键机制。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GABA的影响，包括一系列新的类固醇 对映体和荧光类固醇提供了关于细胞内和 这些药物的膜内蓄积。其中一种荧光类固醇只会增强GABAR 当暴露在可见光(-480 nm)下时，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器。在目前的建议中，我们将延长 我们通过四个目标对神经活性类固醇的研究：1.检测膜和受体 GABA增强3-α-羟基类固醇作用的机制；2.研究 C3-硫酸类固醇和3-β-羟基类固醇影响血管紧张素转换酶的膜机制 GABARs；3.研究光活性类固醇对GABARs影响的机制；以及4. 以获得有关GABAR和NMDAR上类固醇结构要求的新信息。这些 研究将使用生理和细胞成像方法相结合的方法来检查类固醇对 培养的大鼠海马神经元、HEK细胞和非洲爪哇卵母细胞表达特定的GABAR 亚基，并将涉及基于我们最初的结构-活性和机理研究的组合 使用新型荧光和光活性类固醇类似物的观察。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，并更好地理解 类固醇介导麻醉的机制。

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.