Some 25 million patients are given general anesthesia each year in the USA using agents with very low therapeutic indices. The molecular mechanisms of general anesthesia remain unknown, hampering the design of improved agents. General anesthetics are believed to modulate the function of a homologous super-family of postsynaptic ligand-gated ion channels. This PPG focuses on the enhancing action on the inhibitory GABA/A receptor (GABAAR) and the inhibitory action on the excitatory nicotinic receptor, nAcChoR. The overall hypothesis is that general anesthetics bind to a number of sites on these receptors, that their location and affinity varies with the anesthetic's structure and the receptor's conformation, and that parallels exist between the two homologous receptors. The overall aims of the PPG are to: (i) locate anesthetics sites on the GABA/A and nAcCho receptors, and (ii) define their functional significance. The focus is on three regions of the receptors: the extracellular portion, particularly just before the fist transmembrane helix, the second transmembrane helix, and the lipid protein interface. Two complementary techniques will be employed to detect sites, photoaffinity labeling (Projects I, II & IV) and site directed mutagenesis (Projects III and IV). Project II will locate the sites where volatile, alcohol and steroid anesthetics photo-label the equilibrium states of the nAcChoR, and Project IV will use similar techniques, as well as site directed mutagenesis, to locate sites on the GABAAR. Project I will determine which sites inhibit the nAcChoR's open channel using time resolved photo-labeling. Project III will define mechanisms kinetically using rapid perfusion patch clamp techniques in wild type and mutated receptors, incorporating the photo- labeling results to guide mutagenesis and interpretation. Projects I & II will investigate the role of cholesterol sites in modulating allosteric interactions between anesthetic sites and steroid anesthetic action, respectively. Synthetic and Protein Chemistry Cores are essential for developing novel photoaffinity general anesthetics and for locating the sites of photo-incorporation, respectively.
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