title: Role of the histidine residue at position 105 in the human alpha 5 containing GABA(A) receptor on the affinity and efficacy of benzodiazepine site ligands.
abstract: 1. A histidine residue in the N-terminal extracellular region of alpha 1,2,3,5 subunits of the human GABA(A) receptor, which is replaced by an arginine in alpha 4 and alpha 6 subunits, is a major determinant for high affinity binding of classical benzodiazepine (BZ)-site ligands. The effect of mutating this histidine at position 105 in the alpha 5 subunit to an arginine (alpha 5H105R) on BZ-site pharmacology has been investigated using radioligand binding on HEK293 and L(tk-) cells and two electrode voltage clamp recording on Xenopus oocytes in which GABA(A) receptors of subtypes alpha 5, alpha 5H105R, alpha 4 and alpha 6 were co-expressed with beta 3 gamma 2s. 2. The classical BZs, diazepam and flunitrazepam (full agonists on the alpha 5 receptor) showed negligible affinity and therefore negligible efficacy on alpha 5H105R receptors. The beta-carbolines DMCM and beta CCE (inverse agonists on the alpha 5 receptor) retained some affinity but did not exhibit inverse agonist efficacy at alpha 5H105R receptors. Therefore, the alpha 5H105R mutation confers an alpha 4/alpha 6-like pharmacology to the classical BZs and beta-carbolines. 3. Ro15-4513, flumazenil, bretazenil and FG8094, which share a common imidazobenzodiazepine core structure, retained high affinity and were higher efficacy agonists on alpha 5H105R receptors than would be predicted from an alpha 4/alpha 6 pharmacological profile. This effect was antagonized by DMCM, which competes for the BZ-site and therefore is likely to be mediated via the BZ-site. 4. These data indicate that the conserved histidine residue in the alpha subunit is not only a key determinant in the affinity of BZ-site ligands on alpha 5 containing GABA(A) receptors, but also influences ligand efficacy.
authors.affiliation: De Nova Pharmacauticals, St Andrews House, 59 St Andrews Street, Cambridge, CB2 3DD.
authors.forename: M D
authors.initials: MD
authors.lastname: Kelly
authors.forename: A
authors.initials: A
authors.lastname: Smith
authors.forename: G
authors.initials: G
authors.lastname: Banks
authors.forename: P
authors.initials: P
authors.lastname: Wingrove
authors.forename: P W
authors.initials: PW
authors.lastname: Whiting
authors.forename: J
authors.initials: J
authors.lastname: Atack
authors.forename: G R
authors.initials: GR
authors.lastname: Seabrook
authors.forename: K A
authors.initials: KA
authors.lastname: Maubach
mesh_terms: D000927:Anticonvulsants / Q000378:metabolism; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001569:Benzodiazepines / Q000378:metabolism*; D001570:Benzodiazepinones / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002478:Cells, Cultured; D005442:Flumazenil / Q000378:metabolism; D018757:GABA Modulators / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009154:Mutation; D021122:Protein Subunits; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
chemical_list: D000927:Anticonvulsants; D001386:Azides; D001570:Benzodiazepinones; D018757:GABA Modulators; D008024:Ligands; D021122:Protein Subunits; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005442:Flumazenil; D006639:Histidine; D005680:gamma-Aminobutyric Acid; C042957:Ro 15-4513; D001120:Arginine; C054626:bretazenil