title: The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.
abstract: Type A gamma-aminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligand-gated ion channels probably formed from the coassembly of different subunits (alpha 1-6, beta 1-3, gamma 1-3, delta) in the arrangement alpha beta gamma or alpha beta delta. The activation of these receptors by GABA can be modulated by a range of compounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the beta subunit of the GABAA receptor. The action of loreclezole depends on the type of beta subunit present in the receptor complex; receptors containing beta 2 or beta 3 subunits have > 300-fold higher affinity for loreclezole than receptors containing a beta 1 subunit. We have used this property to identify the amino acid residue in the beta subunit that determines the subunit selectivity of loreclezole. Chimeric beta 1/beta 2 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha 1 and gamma 2s subunits. The chimera beta 1/beta 2Lys237-Gly334 conferred sensitivity to 1 microM loreclezole. Within this region there are four amino acids that are conserved in beta 2 and beta 3 but differ in beta 1. By mutating single amino acids of the beta 1 subunit to the beta 2/beta 3 equivalent, only the beta 1 mutation of Ser-290-->Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the beta 2 and beta 3 subunits to the beta 1 equivalent (Asn-->Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors containing the mutated beta subunits. Thus, a single amino acid, beta 2 Asn-289 (beta 3 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.
authors.affiliation: Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom.
authors.forename: P B
authors.initials: PB
authors.lastname: Wingrove
authors.forename: K A
authors.initials: KA
authors.lastname: Wafford
authors.forename: C
authors.initials: C
authors.lastname: Bain
authors.forename: P J
authors.initials: PJ
authors.lastname: Whiting
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology*; D001483:Base Sequence; D017931:DNA Primers; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005260:Female; D005445:Flunitrazepam / Q000494:pharmacology; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014230:Triazoles / Q000494:pharmacology*; D014981:Xenopus; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D000927:Anticonvulsants; D017931:DNA Primers; D018076:DNA, Complementary; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D014230:Triazoles; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam; C066440:loreclezole