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 act on a superfamily of ligand gated channels which include inhibitory anion channels gated by GABA and glycine, and excitatory cation channels gated by serotonin and acetylcholine. This PPG focuses on the ability of general anesthetics to enhance the activity of the inhibitory GABAA receptor (GABAAR) and to inhibit (and in some cases enhance) the excitatory neuronal serotonin (5- HT3R) and nicotinic acetylcholine receptors (nAcChoR). The overall hypothesis is that the various action of general anesthetics are mediated by a number of binding 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 anesthetic binding sites on the GABAA, 5-HT3 and nAcCho receptors, and (ii) define the functional significance of each site. Two complementary techniques will be employed to detect sites, photoaffinity labeling (Projects 2, 3 & 5) and site directed mu:agenesis (Projects 1,3, 4 & 5). Project 1 will characterize the pharmacology of the 5-HT3 receptor and ;nteract with Projects 2 & 3 who will locate the sites of action on activated (time-resolved photolabeling) and desensitized receptor states of alcohols, etomidate and propofol photolabels. Project 4 will define in detail the kinetic mechanisms of anesthetic action on GABAARS using rapid perfusion patch clamp techniques in wild type and mutated receptors, incorporating the photolabeling results to guide mutagenesis and interpretation. Project 5 will locate general anesthetic sites on GABAA receptors using photoactivable general anesthetics. Synthetic and Protein Chemistry Cores are essential for developing novel photoaffinity general anesthetics and for locating the sites of photoincorporation, respectively. A Protein Production Core will supply neuronal receptors to each of the projects.
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