The mechanisms by which general anesthetics have their clinically desired effects are not yet fully understood. The work proposed in this Program is designed to define the molecular target(s) for anesthetic steroids and to clarify the way in which action at these targets results in anesthesia. The underlying idea is that steroid anesthetics act at defined sites on proteins and, in particular, the proteins involved in information transfer between neurons. The proposed research will build on a series of advances made in preceding periods of the Program, which have provided insights into the sites and mechanisms of action for steroids. One objective of the Program is to define the structural basis for steroid interaction with a specific target, the GABA-A receptor. This work will involve complementary studies of the structure-activity relationship for steroid analogues and mutational studies of receptor structure coupled with identification of the residues labeled by site-specific photo-activated steroid analogues. The second objective of the Program is to clarify the mechanisms by which steroids have their actions on their molecular targets, including studies of functional effects on target proteins and examination of steroid access. The third objective is to examine the role of steroid partitioning among cellular membrane pools in determining the time course and magnitude of steroid action. The final objective is to correlate the pharmacological and physiological results with the production of anesthesia or other states defined only at the level of the whole animal. Although a major focus is on studies of the GABA-A receptor, comparative studies of glutamatergic, glycinergic and GABA-C receptors will be performed. Each project addresses one or another aspect of the action of anesthetic steroids at the cellular and molecular level. The Program as a whole will integrate these complementary studies and provide the resources for continued development of novel compounds and for assays of drug effects on behavioral states.
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