This program project seeks to identify the sites of action of general anesthetics on protein targets in the brain, concentrating on the ligand- gated ion channel super-family of neurotransmitter receptors, this project deals with the inhibitory GABA/A receptor-chloride ion channel (GABAR), the major candidate molecular target for the general anesthetics. As with all members of the super-family, GABAR are heteropentamers, comprised of three different sorts of subunits chosen from a repertoire of 18. This results in about two dozen different isoforms which differ in pharmacological properties. Different sensitivity to anesthetics of GABAR of varying subunit composition allow the use of the genetically engineered site-directed chimerae and mutagenesis to identify amino acid residues involved in anesthetic modulation. Others recently have identified several residues in the membrane-spanning domains; we will carry out a series of site-directed mutagenesis studies on the N-terminal extracellular domain of GABAR subunits of varying sensitivity to modulation by anesthetics in order to identify which residues are responsible. More direct identification of anesthetic binding sites can be obtained by photoaffinity labeling with radioactive anesthetics and determination of the amino acid residues that are covalently bound by microsequencing. This study will employ recombinant GABAR expressed in the insect Sf9 cell line using Baculovirus. In particular we will develop a new radioactive anesthetic steroid photoaffinity label for the GABAR work. The importance of labeled residues will be verified by mutagenesis and analysis of anesthetic sensitivity using binding and electrophysiology. The combination of these approaches, and comparison with sites of interaction in other members of the receptor super-family especially nicotinic acetylcholine receptors, will lead to a greater understanding of the mechanism of action of these clinically important drugs.
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