Extensive heterogeneity of recombinant gamma-aminobutyric acidA receptors expressed in alpha 4 beta 3 gamma 2-transfected human embryonic kidney 293 cells.
Human embryonic kidney 293 cells transiently transfected with alpha 4-, beta 3- and gamma 2-subunits of gamma-aminobutyric acidA (GABAA) receptors from the rat exhibited specific high affinity binding sites for [3H]muscimol, [3H]Ro 15-4513 and [35S]t-butylbicyclophosphorothionate (TBPS). Bmax values obtained, however, were dramatically different for these compounds. In addition, GABA was able to inhibit only 20% of specific [35S]TBPS binding to membranes from alpha 4 beta 3 gamma 2-transfected cells. In order to investigate possible receptor heterogeneity, receptors were extracted from alpha 4 beta 3 gamma 2-transfected cells and were fractionated by chromatography on an anti-gamma 2-, followed by an anti-alpha 4- and an anti-beta 3-immunoaffinity column. Western blot analysis of the column eluates indicated the separate existence of GABAA receptors consisting of alpha 4 beta 3 gamma 2-, alpha 4 beta 3- or beta 3-subunits in alpha 4 beta 3 gamma 2-transfected cells. This, and the finding that only alpha 4 beta 3 gamma 2- but not alpha 4 beta 3- or beta 3-receptors possess high affinity binding sites for all three radiolabeled ligands investigated, combined with the observation that [35S]TBPS binding to receptors consisting of beta 3-subunits cannot be inhibited by GABA, can explain most of the binding data obtained. The present results suggest an inefficient assembly of gamma 2- with alpha 4- and/or beta 3-subunits under the conditions used, and indicate that recombinant receptors expressed in HEK cells are not necessarily homogeneous.
Ebert
V
V
Department of Biochemical Psychiatry, University Clinic for Psychiatry, Vienna, Austria.
Scholze
P
P
Sieghart
W
W
D000345:Affinity Labels; D001386:Azides / Q000378:metabolism; D001569:Benzodiazepines / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D018755:GABA Agonists / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009118:Muscimol / Q000378:metabolism; D011963:Receptors, GABA-A / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D000345:Affinity Labels; D001386:Azides; D018755:GABA Agonists; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D009118:Muscimol; C042957:Ro 15-4513