Molecular and pharmacological characterization of native cortical gamma-aminobutyric acid(A) receptors containing both alpha(1) and alpha(3) subunits

We have investigated the existence, molecular composition, and benzodiazepine binding properties of native cortical alpha(1)-alpha(3) gamma-aminobutyric acid(A) (GABA(A)) receptors using subunit-specific antibodies. The co-existence of alpha(1) and alpha(3) subunits in native GABA(A) receptors was demonstrated by immunoblot analysis of the anti-alpha(1)- or anti-alpha(3)-immunopurified receptors and by immunoprecipitation experiments of the [H-3]zolpidem binding activity. Furthermore, immunodepletion experiments indicated that the alpha(1)-alpha(3) GABA(A) receptors represented 54.7 +/- 5.0 and 23.6 +/- 3.3% of the alpha(3) and alpha(1) populations, respectively. Therefore, alpha(1) and alpha(3) subunits are associated in the same native GABA(A) receptor complex, but, on the other hand, these alpha(1)-alpha(3) GABA(A) receptors from the cortex constitute a large proportion of the total alpha(3) population and a relatively minor component of the alpha(1) population. The pharmacological analysis of the alpha(1)- or alpha(3)-immunopurified receptors demonstrated the presence of two different benzodiazepine binding sites in each receptor population with high (type I binding sites) and low (type II binding sites) affinities for zolpidem and CI 218,872. These results indicate the existence of native GABA(A) receptors possessing both alpha(1) and alpha(3) subunits, with alpha(1) and ag subunits expressing their characteristic benzodiazepine pharmacology. The molecular characterization of the anti-alpha(1)-anti-alpha(3) double-immunopurified receptors demonstrated the presence of stoichiometric amounts of alpha(1) and alpha(3) subunits, associated with beta(2/3), and gamma(2) subunits. The pharmacological analysis of alpha(1)-alpha(3) GABA(A) receptors demonstrated that, despite the fact that each alpha subunit retained its benzodiazepine binding properties, the relative proportion between type I and II binding sites or between 51- and 59-61-kDa [H-3]Ro15-4513-photolabeled peptides was 70:30. Therefore, the alpha(1) subunit is pharmacologically predominant over the alpha(3) subunit. These results indicate the existence of active and nonactive alpha subunits in the native alpha(1)-alpha(3) GABA(A) receptors from rat cortex.