Abstract
Evidences indicate the existence of two homologous and/or heterologous α subunits coassembled in a single γ-aminobutyric acid type A (GABAA) receptor. However, it is unknown whether both or only one of the coassembled α subunits display benzodiazepine binding sites. Thus, we have investigated the association between α1 and α5 subunits and the pharmacological properties of these GABAA receptors from rat hippocampus. The association between α1 and α5 subunits was demonstrated by immunoblot of the anti-α1 or -α5 immunoaffinity-purified receptors and by double immunopurification by anti-α1 and -α5 columns in series. The benzodiazepine binding properties of the immunoprecipitated receptors indicated the existence of pharmacologically active and inactive α subunits. The anti-α5 immunoprecipitated receptors displayed exclusively low-affinity binding sites for both Cl218,872 (Ki = 0.81 ± 0.15 μM) and zolpidem (Ki = 5.0 ± 3.0 μM), in spite of the association between α1 and α5 subunits. The anti-α1 immunoprecipitated receptors displayed both high- and low-affinity binding sites for both ligands (Kis = 47.5 ± 5.2 nM and 0.7 ± 0.06 μM for Cl218,872 and 25.0 ± 7.0 nM, 415 ± 200 nM and 9.3 ± 3.0 μM for zolpidem). Therefore, the α5 subunit, when coassembled with α1 subunit, should be pharmacologically predominant. This hypothesis was probed by immunoprecipitation of the photoaffinity-labeled receptors and by anti-α1 and -α5 double immunopurified receptors. The α1-α5 double immunopurified receptors displayed a single low-affinity binding site (Ki = 908 ± 105 nM) for Cl218,872, undetectable [3H]zolpidem binding activity, and similar [3H]flumazenil and [3H]L-655,708 binding activity (0.10 ± 0.01 and 0.09 ± 0.02 pmol/20 μl of anti-α5 immunobeads, respectively). Thus, the native GABAA receptors containing α1 and α5 subunits have only one α subunit pharmacologically active displaying α5 binding properties.
Footnotes
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Send reprint requests to: J. Vitorica, Departamento de Bioquı́mica, Bromatologı́a y Toxicologı́a, Facultad de Farmacia, Universidad de Sevilla, 41012, Sevilla, Spain. E-mail: vitorica{at}cica.es
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↵1 This work was supported by Grants PB93-0739 from Direccion General de Investigacion Cientifica y Tecnica and 97/1303 from Fondo de Investigaciones Sanitarias. D.R. is supported by a contract from the Ministerio de Educación y Cultura.
- Abbreviations:
- GABA
- γ-aminobutyric acid
- FMZ
- flumazenil
- Received February 11, 1999.
- Accepted April 30, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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