General Anesthetic Sites on Ligand-Gated Ion Channels

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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 nicotinic acetylcholine (nAcChoR) and serotonin (5HT3R) receptors. The overall hypothesis is that the various actions 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. The overall aims of the PPG are to: (i) locate the anesthetic binding sites on the GABAA, nAcChoR and 5-HT3 receptors using anesthetic photolabels, and (ii) define how their occupancy allosterically modulates receptor function using rapid perfusion patch clamp techniques. Project 1 will locate sites of etomidate, propofol and barbiturate photolabels on equilibrium states of GABAAR and nAcChoRs. Project 2 focuses on the interaction of anesthetics with receptors during gating using in parallel electrophysiological and time-resolved photolabeling. Project 3 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. A Synthetic Chemistry Core develops and supplies novel photoaffinity general anesthetics. A Protein Chemistry Core locates the sites of photoincorporation and develops homology models of receptors to guide mutagenesis work. A Protein Production Core supplies large quantities of heterologously expressed, purified and reconstituted neuronal receptors for photolabeling.