Vol. 290, Issue 3, 989-997, September 1999

Native -Aminobutyric Acid Type A Receptors from Rat Hippocampus, Containing Both 1 and 5 Subunits, Exhibit a Single Benzodiazepine Binding Site with 5 Pharmacological Properties¹

Francisco Araujo, Diego Ruano and Javier Vitorica

Department of Bioquimica, Bromatologia y Toxicologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain

Evidences indicate the existence of two homologous and/or heterologous  subunits coassembled in a single -aminobutyric acid type A (GABA_A) receptor. However, it is unknown whether both or only one of the coassembled  subunits display benzodiazepine binding sites. Thus, we have investigated the association between 1 and 5 subunits and the pharmacological properties of these GABA_A receptors from rat hippocampus. The association between 1 and 5 subunits was demonstrated by immunoblot of the anti-1 or -5 immunoaffinity-purified receptors and by double immunopurification by anti-1 and -5 columns in series. The benzodiazepine binding properties of the immunoprecipitated receptors indicated the existence of pharmacologically active and inactive  subunits. The anti-5 immunoprecipitated receptors displayed exclusively low-affinity binding sites for both Cl218,872 (K_i = 0.81 ± 0.15 µM) and zolpidem (K_i = 5.0 ± 3.0 µM), in spite of the association between 1 and 5 subunits. The anti-1 immunoprecipitated receptors displayed both high- and low-affinity binding sites for both ligands (K_is = 47.5 ± 5.2 nM and 0.7 ± 0.06 µM for Cl218,872 and 25.0 ± 7.0 nM, 415 ± 200 nM and 9.3 ± 3.0 µM for zolpidem). Therefore, the 5 subunit, when coassembled with 1 subunit, should be pharmacologically predominant. This hypothesis was probed by immunoprecipitation of the photoaffinity-labeled receptors and by anti-1 and -5 double immunopurified receptors. The 1-5 double immunopurified receptors displayed a single low-affinity binding site (K_i = 908 ± 105 nM) for Cl218,872, undetectable [³H]zolpidem binding activity, and similar [³H]flumazenil and [³H]L-655,708 binding activity (0.10 ± 0.01 and 0.09 ± 0.02 pmol/20 µl of anti-5 immunobeads, respectively). Thus, the native GABA_A receptors containing 1 and 5 subunits have only one  subunit pharmacologically active displaying 5 binding properties.