Project 1: Locating General Anesthetic Binding Sites in GABAA & Acetylcholine Rec

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LOCATING GENERAL ANESTHETIC BINDING SITES IN GABAA AND ACETYLCHOLINE RECEPTORS Our goal is to determine the number and location of the binding sites for general anesthetics in GABAARS, a major site of action of a structurally diverse group of general anesthetics used clinically, and to compare them with their binding sites in nicotinic acetylcholine receptors (AcChoRs), receptors that are both members of the "Cys-loop" superfamily of neurotransmitter-gated ion channels. Most anesthetics potentiate the action of GABA at the inhibitory GABAA receptors, while they inhibit the excitatory AcChoRs in brain and skeletal muscle. Within a single receptor multiple potential anesthetic binding sites are present in the pockets that exist within receptor subunits and at subunit interfaces, including the ion channel. It is our hypothesis that each anesthetic, depending on structural class, will interact preferentially with different receptor sites. An improved understanding of the diversity of general anesthetic binding sites within neurotransmitter-gated ion channels will allow for the design of safer anesthetic agents. We propose to use photoaffinity labeling and protein chemistry techniques to identify the binding sites for photoreactive aliphatic alcohols and analogs of etomidate, propofol, and barbiturates in GABAARS isolated from detergent extracts of bovine brain and cultured cells, in neuronal a4p2 AcChoRs isolated from cultured cells, and in muscle-type AcChoRs in nicotinic postsynaptic membranes isolated from Torpedo electric organ. We will determine whether a single anesthetic binding site in a GABAAR (or AcChoR) can be occupied by drugs that, depending on structure, act as potentiators or inhibitors. These photoaffinity labeling studies make use of the Synthetic Chemistry, Protein Chemistry and Protein Production Cores. Our structural studies are complementary to the time- resolved photolabeling studies in (Miller/Raines Project) and to the mutational analyses (Forman Project) designed to determine whether the sites we identify are sites of functional potentiation or inhibition. RELEVANCE (See instructions): GABAARS in the brain are a major site of action of many clinically used anesthetics of diverse chemical structure, but the number and location of anesthetic binding sites within GABAARS or in the structurally related nicotinic acetylcholine receptors remains unknown. Identification of these sites will contribute to the development of anesthetics with greater selectivity and fewer side-effects.