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
F. Araujo et al., 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, J PHARM EXP, 290(3), 1999, pp. 989-997
Citations number
35
Categorie Soggetti
Pharmacology & Toxicology
Journal title
JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
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.