title: Rat and human hippocampal alpha5 subunit-containing gamma-aminobutyric AcidA receptors have alpha5 beta3 gamma2 pharmacological characteristics.
abstract: The gamma-aminobutyric acid (GABA)A receptor is a hetero-oligomer consisting of five subunits, the combination of which confers unique pharmacological properties to the receptor. To understand the physiological role of native GABAA receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human alpha5 beta3 gamma2 and alpha5beta3gamma3 GABAA receptors stably expressed in L(tk-) cells were characterized with the alpha5-selective ligand [3H]L-655,708 and compared with the pharmacological characteristics of [3H]L-655,708 binding sites from rat and human hippocampus. Saturation analyses revealed a 9-fold selective affinity of [3H]L-655,708 for alpha5 beta3 gamma2 receptors (Kd = 1.7 +/- 0.4 nM), compared with alpha5 beta3 gamma3 receptors (Kd = 15 +/- 3 nM). Rat and human hippocampal [3H]L-655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the affinity of alpha5 beta3 gamma2 receptors. Pharmacological analysis of [3H]L-655,708 binding sites in rat and human hippocampi revealed a strong correlation with the affinities of seven benzodiazepine site ligands for alpha5 beta3 gamma2 but not alpha5 beta3 gamma3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a gamma2-selective antibody yielded 19 +/- 4% of total benzodiazepine binding sites measured using [3H]Ro15-1788, whereas no specific binding was measured after immunoprecipitation with an anti-gamma3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-alpha5 and anti-gamma2 or anti-alpha5 and anti-gamma3 antibodies established the preferential expression of alpha5 gamma2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of alpha5 beta3 gamma2 GABAA receptors in rat hippocampus, despite the clustering of alpha5 and gamma3 loci on the same chromosome.
authors.affiliation: Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, CM20 2QR, UK. crrille_sur@merck.com
authors.forename: C
authors.initials: C
authors.lastname: Sur
authors.forename: K
authors.initials: K
authors.lastname: Quirk
authors.forename: D
authors.initials: D
authors.lastname: Dewar
authors.forename: J
authors.initials: J
authors.lastname: Atack
authors.forename: R
authors.initials: R
authors.lastname: McKernan
mesh_terms: D000328:Adult; D000818:Animals; D001667:Binding, Competitive; D006624:Hippocampus / Q000648:ultrastructure*; D006801:Humans; D007093:Imidazoles / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D046911:Macromolecular Substances; D008297:Male; D011233:Precipitin Tests; D011487:Protein Conformation; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D014316:Tritium
chemical_list: D007093:Imidazoles; C105490:L 655,708; D008024:Ligands; D046911:Macromolecular Substances; D011963:Receptors, GABA-A; D014316:Tritium