Analysis of the presence and abundance of GABA_A receptors containing two different types of α subunits in murine brain using point-mutated α subunits

γ-Aminobutyric acid, type A (GABA_A) receptors are pentameric proteins of which the majority is composed of two α subunits, two β subunits and one γ subunit. It is well documented that two different types of α subunits can exist in a singles GABA_A receptor complex. However, information on the abundance of such GABA_A receptors is rather limited. Here we tested whether mice containing the His to Arg point mutation in the α1, α2, or α3 subunit at positions 101, 101, and 126, respectively, which render the respective subunits insensitive to diazepam, would be suitable to analyze this issue. Immunodepletion studies indicated that the His to Arg point mutation solely rendered those GABA_A receptors totally insensitive to diazepam binding that contain two mutated α subunits in the receptor complex, whereas receptors containing one mutated and one heterologous α subunit not carrying the mutation remained sensitive to diazepam binding. This feature permitted a quantitative analysis of native GABA_A receptors containing heterologous α subunits by comparing the diazepam-insensitive binding sites in mutant mouse lines containing one mutated α subunit with those present in mouse lines containing two different mutated α subunits. The data indicate that the α1α1-containing receptors with 61% is the most abundant receptor subtype in brain, whereas the α1α2 (13%), α1α3 (15%), α2α2 (12%), α2α3 (2%), and α3α3 combinations (4%) are considerably less expressed. Only within the α1-containing receptor population does the combination of equal α subunits (84% α1α1, 7% α1α2, and 8% α1α3) prevail, whereas in the α2-containing receptor population (46% α2α2, 36% α2α1, and 19% α2α3) and particularly in the α3-containing receptor population (27% α3α3, 56% α3α1, and 19% α3α2), the receptors with two different types of α subunits predominate. This experimental approach provides the basis for a detailed analysis of the abundance of GABA_A receptors containing heterologous α subunits on a brain regional level.