Absence of association between delta and gamma2 subunits in native GABA(A) receptors from rat brain

Abstract

We investigated the possible association between delta and gamma2 subunits in native GABA(A) receptors, from different rat brain regions, using subunit-specific anti-delta and anti-gamma2 antibodies. Previous reports have provided somewhat controversial results, indicating both the presence and the absence of association between these two subunits in native receptors. Our results indicate the absence of co-localization between delta and gamma2 subunits. In immunoprecipitation experiments, anti-delta antibody consistently immunoprecipitated [3H]muscimol binding activity (GABA binding sites) from all brain areas tested (10-20% of the total binding). However, under the same conditions, no significant [3H]flumazenil or [3H]ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazol[1,5-a]-[1,4]benzodiazepine- 3-carboxylate (Ro15-4513) binding (benzodiazepine binding sites) activity was detected in the immunopellets. These results indicate the absence of association between delta and gamma2 subunits. This question was directly addressed by immunopurification and Western blot experiments. As expected, no gamma2 subunits were detected in anti-delta immunoaffinity purified receptors. Conversely, no delta subunits were identified in anti-gamma2 immunopurified receptors. Thus, these results demonstrate the absence of association between delta and gamma2 subunits in native GABA(A) receptors. Finally, our results also indicate the relevance of the solubilization conditions on the apparent association between different subunits of the native GABA(A) receptor complex.