Rat and Human Hippocampal α5 Subunit-Containing γ-Aminobutyric AcidA Receptors Have α5β3γ2 Pharmaco logical Characteristics

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Abstract
The γ-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 GABAAreceptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human α5β3γ2 and α5β3γ3 GABAA receptors stably expressed in L(tk−) cells were characterized with the α5-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 α5β3γ2 receptors (Kd = 1.7 ± 0.4 nm), compared with α5β3γ3 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 α5β3γ2 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 α5β3γ2 but not α5β3γ3 receptors. Immunoprecipitation of [3H]L-655,708 binding sites from rat hippocampus with a γ2-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-γ3 antibody. Combinatorial immunoprecipitations of [3H]muscimol binding sites with anti-α5 and anti-γ2 or anti-α5 and anti-γ3 antibodies established the preferential expression of α5γ2 receptors, accounting for 22 ± 2% of total rat hippocampal GABAA receptors. These observations provide pharmacological and structural evidence for the prevalence of α5β3γ2 GABAA receptors in rat hippocampus, despite the clustering of α5 and γ3 loci on the same chromosome.