Native gamma-aminobutyric acid type A receptors from rat hippocampus, containing both alpha 1 and alpha 5 subunits, exhibit a single benzodiazepine binding site with alpha 5 pharmacological properties

Evidences indicate the existence of two homologous and/or heterologous cu s ubunits coassembled in a single gamma-aminobutyric acid type A (GABA(A)) re ceptor. However, it is unknown whether both or only one of the coassembled cw subunits display benzodiazepine binding sites. Thus, we have investigate d the association between alpha 1 and alpha 5 subunits and the pharmacologi cal properties of these GABA(A) receptors from rat hippocampus. The associa tion between alpha 1 and alpha 5 subunits was demonstrated by immunoblot of the anti-alpha 1 or -alpha 5 immunoaffinity-purified receptors and by doub le immunopurification by anti-al and -alpha 5 columns in series. The benzod iazepine binding properties of the immunoprecipitated receptors indicated t he existence of pharmacologically active and inactive or subunits. The anti -alpha 5 immunoprecipitated receptors displayed exclusively low-affinity bi nding sites for both Cl218,872 (K-i = 0.81 +/- 0.15 mu M) and zolpidem (K-i = 5.0 +/- 3.0 mu M), in spite of the association between alpha 1 and alpha 5 subunits. The anti-arl immuno-precipitated receptors displayed both high - and low-affinity binding sites for both ligands (K(i)s = 47.5 +/- 5.2 nM and 0.7 +/- 0.06 mu M for Cl218,872 and 25.0 +/- 7.0 nM, 415 +/- 200 nM and 9.3 +/- 3.0 mu M for zolpidem). Therefore, the alpha 5 subunit, when coass embled with oil subunit, should be pharmacologically predominant. This hypo thesis was probed by immunoprecipitation of the photoaffinity-labeled recep tors and by anti-oil and -alpha 5 double immunopurified receptors. The alph a 1-alpha 5 double immunopurified receptors displayed a single low-affinity binding site (K-i = 908 +/- 105 nM) for Cl218,872, undetectable [H-3]zolpi dem binding activity, and similar [H-3]flumazenil and [H-3]L-655,708 bindin g activity (0.10 +/- 0.01 and 0.09 +/- 0.02 pmol/20 mu l of anti-alpha 5 im munobeads, respectively). Thus, the native GABA(A) receptors containing alp ha 1 and alpha 5 subunits have only one oc subunit pharmacologically active displaying a5 binding properties.