This project will examine the actions of steroid anesthetics on transmitter-gatedmembrane channels. The experiments are designed to determine the sites on receptors to which the steroids bind, and define the mechanism(s) by which functional effects are mediated. The main focus of the proposed work is the gamma-amino butyric acid type-A receptor (GABA-A receptor), which has been proposed as a major site of action for general anesthetics, including steroids. Defined combinations of subunits will be expressed in non-neural cells. Molecular biological techniques will be used to manipulate the structure of the expressed subunits, to define regions of GABA-A receptor subunits required for anesthetic action. Complementary work will use pharmacological and biophysical approachers to examine the mechanism for anesthetic action. Stable clonal cell lines expressing high levels of GABA-A receptors will be generated for use in physiological and biochemical studies. The studies of GABA-A receptors will be extended by comparative data obtained for anesthetic actions on the major brain nicotinic receptor, stably expressed in non- neural cells. Finally, the actions of the intravenous anesthetics pentobarbital and propofol will be compared to those of steroids in these experiments. The results should lead to insights into the mechanisms by which steroid anesthetics act at one molecular target, the GABA-A receptors. Comparisons with other data obtained in this project and the Program as a whole will indicate the range of effects which anesthetics have on several identified membrane channels involved in synaptic transmission. This information is required for a better understanding of the cellular actions associated with anesthesia.
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