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

Type A gamma-aminobutyric acid (GABA(A)) 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 GABA(A) 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 GABA(A) receptor subunits were constructed and coexpressed in Xenopus oocytes with human alpha(1) and gamma(2s), subunits. The chimera beta(1)/beta(2)Lys237-Gly334 conferred sensitivity to 1 mu M 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.