RAT AND HUMAN HIPPOCAMPAL ALPHA-5 SUBUNIT-CONTAINING GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS HAVE ALPHA-5-BETA-3-GAMMA-2 PHARMACOLOGICAL CHARACTERISTICS

The gamma-aminobutyric acid (GABA)(A) receptor is a hetero-oligomer co nsisting of five subunits, the combination of which confers unique pha rmacological properties to the receptor. To understand the physiologic al role of native GABA(A) receptors, it is critical to determine their subunit compositions. The pharmacological characteristics of human al pha 5 beta 3 gamma 2 and alpha 5 beta 3 gamma 3 GABA(A) receptors stab ly expressed in L(tk(-)) cells were characterized with the alpha 5-sel ective ligand [H-3]L-655,708 and compared with the pharmacological cha racteristics of [H-3]L-655,708 binding sites from rat and human hippoc ampus. Saturation analyses revealed a 9-fold selective affinity of [H- 3]L-655,708 for alpha 5 beta 3 gamma 2 receptors (K-d = 1.7 +/- 0.4 nM ), compared with alpha 5 beta 3 gamma 3 receptors (K-d = 15 +/- 3 nM). Rat and human hippocampal [H-3]L655,708 binding sites had affinities of 2.2 +/- 0.6 and 1.0 +/- 0.2 nM, respectively, comparable to the aff inity of alpha 5 beta 3 gamma 2 receptors. Pharmacological analysis of [H-3]L-655,708 binding sites in rat and human hippocampi revealed a s trong correlation with the affinities of seven benzodiazepine site lig ands for alpha 5 beta 3 gamma 2 but not alpha 5 beta 3 gamma 3 recepto rs. Immunoprecipitation of [H-3]L-655,708 binding sites from rat hippo campus with a gamma 2-selective antibody yielded 19 +/- 4% of total be nzodiazepine binding sites measured using [H-3]Ro15-1788, whereas no s pecific binding was measured after immunoprecipitation with an anti-ga mma 3 antibody. Combinatorial immunoprecipitations of [H-3]muscimol bi nding sites with anti-alpha 5 and anti-gamma 2 or anti-alpha 5 and ant i-gamma 3 antibodies established the preferential expression of alpha 5 gamma 2 receptors, accounting for 22 +/- 2% of total rat hippocampal GABA(A) receptors. These observations provide pharmacological and str uctural evidence for the prevalence of alpha 5 beta 3 gamma 2 GABA(A) receptors in rat hippocampus, despite the clustering of alpha 5 and ga mma 3 loci on the same chromosome.