Identification of GABA(A) receptor regulatory domains by photoaffinity labeling, microsequencing and peptide mapping

The GABA{dollar}sb{lcub}rm A{rcub}{dollar} receptor is a multimeric protein which is the primary target for the major inhibitory neurotransmitter GABA in the mammalian brain. This receptor complex contains a number of modulatory sites of pharmacological and clinical importance. These include sites for benzodiazepines, barbiturates, anesthetic steroids, ethanol, and certain convulsants. The structure of the receptor protein is thought to be similar to that of other members of the ligand gated ion channel superfamily of receptors, to which the GABA receptor belongs. While progress has been made in elucidating domains of the receptor protein which may form these modulatory sites in other members of the superfamily, particularly nicotinic acetylcholine receptors, such information has not been forthcoming for GABA{dollar}sb{lcub}rm A{rcub}{dollar} receptors.; The present study sought to investigate possible protein domains of the receptor which interact with ligands. In particular, it was hypothesized that ligands which bind to the GABA agonist site would interact with a domain of the protein exposed to extracellular water, due to the polar nature of the amino acid ligand. In contrast, ligands which bind to the benzodiazepine site are lipid soluble, and were hypothesized to bind to a hydrophobic domain of the receptor, possibly within a cell membrane spanning domain.; The technique of photoaffinity labeling with tritiated ligands was used combined with peptide mapping and microsequencing strategies to identify amino acids which were specifically labeled in these two binding sites. In addition, similar mapping and sequencing techniques were used to identify regions of the receptor phosphorylated by cyclic AMP-dependent protein kinase (PKA).; The intracellular domain of the receptor {dollar}beta{dollar} subunit which contains the consensus sequence for PKA phosphorylation was identified by sequencing. An amino acid, Phe 65, of the bovine receptor {dollar}alpha{dollar} subunit were identified which was modified by the GABA site agonist label ({dollar}sp3{dollar}H) muscimol. A second domain of the {dollar}alpha{dollar} subunit was tentatively identified as contributing to the benzodiazepine binding site.; These results are discussed as they pertain to the pharmacology of GABA{dollar}sb{lcub}rm A{rcub}{dollar} receptors. In addition, a model of ligand binding to this superfamily of receptors is suggested based upon the results presented.