Araujo F, Ruano D, Vitorica J
Department of Bioquimica, Bromatologia y Toxicologia, Facultad de Farmacia, Universidad de Sevilla, Sevilla, Spain.
J Pharmacol Exp Ther. 1999 Sep;290(3):989-97.
Evidences indicate the existence of two homologous and/or heterologous alpha subunits coassembled in a single gamma-aminobutyric acid type A (GABA(A)) receptor. However, it is unknown whether both or only one of the coassembled alpha subunits display benzodiazepine binding sites. Thus, we have investigated the association between alpha1 and alpha5 subunits and the pharmacological properties of these GABA(A) receptors from rat hippocampus. The association between alpha1 and alpha5 subunits was demonstrated by immunoblot of the anti-alpha1 or -alpha5 immunoaffinity-purified receptors and by double immunopurification by anti-alpha1 and -alpha5 columns in series. The benzodiazepine binding properties of the immunoprecipitated receptors indicated the existence of pharmacologically active and inactive alpha subunits. The anti-alpha5 immunoprecipitated receptors displayed exclusively low-affinity binding sites for both Cl218,872 (K(i) = 0.81 +/- 0.15 microM) and zolpidem (K(i) = 5.0 +/- 3.0 microM), in spite of the association between alpha1 and alpha5 subunits. The anti-alpha1 immunoprecipitated 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 microM for Cl218,872 and 25.0 +/- 7.0 nM, 415 +/- 200 nM and 9. 3 +/- 3.0 microM for zolpidem). Therefore, the alpha5 subunit, when coassembled with alpha1 subunit, should be pharmacologically predominant. This hypothesis was probed by immunoprecipitation of the photoaffinity-labeled receptors and by anti-alpha1 and -alpha5 double immunopurified receptors. The alpha1-alpha5 double immunopurified receptors displayed a single low-affinity binding site (K(i) = 908 +/- 105 nM) for Cl218,872, undetectable [(3)H]zolpidem binding activity, and similar [(3)H]flumazenil and [(3)H]L-655,708 binding activity (0.10 +/- 0.01 and 0.09 +/- 0.02 pmol/20 microliters of anti-alpha5 immunobeads, respectively). Thus, the native GABA(A) receptors containing alpha1 and alpha5 subunits have only one alpha subunit pharmacologically active displaying alpha5 binding properties.
有证据表明，在单个A型γ-氨基丁酸（GABA(A)）受体中存在两个同源和/或异源α亚基共同组装的情况。然而，尚不清楚共同组装的α亚基是两个都显示苯二氮䓬结合位点，还是只有一个显示。因此，我们研究了α1和α5亚基之间的关联以及大鼠海马体中这些GABA(A)受体的药理学特性。通过抗α1或α5免疫亲和纯化受体的免疫印迹以及通过抗α1和α5柱串联的双重免疫纯化，证明了α1和α5亚基之间的关联。免疫沉淀受体的苯二氮䓬结合特性表明存在药理活性和无活性的α亚基。尽管α1和α5亚基之间有关联，但抗α5免疫沉淀受体对氯氮卓（Cl218,872）（K(i)=0.81±0.15微摩尔）和唑吡坦（K(i)=5.0±3.0微摩尔）均仅显示低亲和力结合位点。抗α1免疫沉淀受体对两种配体均显示高亲和力和低亲和力结合位点（氯氮卓的K(i)分别为47.5±5.2纳摩尔和0.7±0.06微摩尔，唑吡坦的K(i)分别为25.0±7.0纳摩尔、415±200纳摩尔和9.3±3.0微摩尔）。因此，当与α1亚基共同组装时，α5亚基在药理学上应占主导地位。通过光亲和标记受体的免疫沉淀和抗α1和α5双重免疫纯化受体对这一假设进行了探究。α1-α5双重免疫纯化受体对氯氮卓显示单一低亲和力结合位点（K(i)=908±105纳摩尔），未检测到[(3)H]唑吡坦结合活性，并且[(3)H]氟马西尼和[(3)H]L-655,708结合活性相似（分别为0.10±0.01和0.09±0.02皮摩尔/20微升抗α5免疫珠）。因此，含有α1和α5亚基的天然GABA(A)受体只有一个具有药理学活性的α亚基，显示α5结合特性。
