PMID- 8183949 OWN - NLM STAT- MEDLINE DCOM- 19940616 LR - 20190501 IS - 0027-8424 (Print) IS - 1091-6490 (Electronic) IS - 0027-8424 (Linking) VI - 91 IP - 10 DP - 1994 May 10 TI - 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. PG - 4569-73 AB - 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. FAU - Wingrove, P B AU - Wingrove PB AD - Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom. FAU - Wafford, K A AU - Wafford KA FAU - Bain, C AU - Bain C FAU - Whiting, P J AU - Whiting PJ LA - eng PT - Journal Article PL - United States TA - Proc Natl Acad Sci U S A JT - Proceedings of the National Academy of Sciences of the United States of America JID - 7505876 RN - 0 (Anticonvulsants) RN - 0 (DNA Primers) RN - 0 (DNA, Complementary) RN - 0 (Macromolecular Substances) RN - 0 (Receptors, GABA-A) RN - 0 (Recombinant Fusion Proteins) RN - 0 (Triazoles) RN - 56-12-2 (gamma-Aminobutyric Acid) RN - 620X0222FQ (Flunitrazepam) RN - 6DJ32STZ5W (loreclezole) SB - IM MH - Allosteric Site MH - Amino Acid Sequence MH - Animals MH - Anticonvulsants/*pharmacology MH - Base Sequence MH - DNA Primers MH - DNA, Complementary/metabolism MH - Dose-Response Relationship, Drug MH - Female MH - Flunitrazepam/pharmacology MH - Humans MH - Macromolecular Substances MH - Molecular Sequence Data MH - Mutagenesis, Site-Directed MH - Oocytes/drug effects/physiology MH - Point Mutation MH - Polymerase Chain Reaction MH - Receptors, GABA-A/chemistry/drug effects/*physiology MH - Recombinant Fusion Proteins/chemistry/drug effects/metabolism MH - Triazoles/*pharmacology MH - Xenopus MH - gamma-Aminobutyric Acid/*pharmacology PMC - PMC43827 EDAT- 1994/05/10 00:00 MHDA- 1994/05/10 00:01 PMCR- 1994/11/10 CRDT- 1994/05/10 00:00 PHST- 1994/05/10 00:00 [pubmed] PHST- 1994/05/10 00:01 [medline] PHST- 1994/05/10 00:00 [entrez] PHST- 1994/11/10 00:00 [pmc-release] AID - 10.1073/pnas.91.10.4569 [doi] PST - ppublish SO - Proc Natl Acad Sci U S A. 1994 May 10;91(10):4569-73. doi: 10.1073/pnas.91.10.4569.