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 inthe absence of GABA at higher concentrations. Other steroids, particularly those with charged substituentsat 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 inhibitionrepresent two key mechanisms in clinical anesthesia. Over the several years, we have developed tools thathave helped us to probe the effects of neuroactive steroids on GABARs, including a series of novel steroidenantiomers and fluorescent steroids that have provided important new information about intracellular andintramembranous accumulation of these agents. One of the fluorescent steroids only potentiates GABARswhen exposed to visible (-480 nm) light, offering the potential to develop a new class of GABAergicmodulators that can be activated in specific regions of cells or brain. In the present proposal, we will extendour studies of neuroactive steroids by addressing four goals: 1. To examine membrane and receptormechanisms contributing to the effects of GABA potentiating 3-alpha-hydroxysteroids; 2. To examinemembrane mechanisms contributing to the effects of C3-sulfated steroids and 3-beta-hydroxysteroids onGABARs; 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. Thesestudies will use a combination of physiological and cellular imaging methods to examine steroid effects incultured rat hippocampal neurons, and HEK cells and Xenopus oocytes expressing defined GABARsubunits, and will involve a combination of structure-activity and mechanistic studies based upon our initialobservations with the novel fluorescent and photoactive steroid analogues. These studies have the potentialto provide new information about neuroactive steroid effects in the CMS and a better understanding ofmechanisms involved in steroid-mediated anesthesia.
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