title: Chloride channel expression with the tandem construct of alpha 6-beta 2 GABAA receptor subunit requires a monomeric subunit of alpha 6 or gamma 2.
abstract: Despite the presence of the multiple subunits (alpha, beta, gamma, and delta) and their isoforms for gamma-aminobutyric acid, type A (GABAA) receptors in mammalian brains, the alpha x beta 2 gamma 2 subtypes appear to be the prototype GABAA receptors sharing many properties with native neuronal receptors. In order to gain insight into their subunit stoichiometry and orientation, we prepared a tandem construct of the alpha 6 and beta 2 subunit cDNAs where the carboxyl-terminal of alpha 6 is linked to the amino-terminal of beta 2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cells with the tandem construct alone failed to induce GABA-dependent CI- currents, but its cotransfection with the cDNA for alpha 6 or gamma 2, but not beta 2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of gamma 2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [3H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the beta 2. No appreciable differences were found in their Kd (2.5 nM) and Bmax values (1.4 pmol/mg of protein). These data are consistent with the view that the polypeptides arising from the tandem construct were expressed with the high affinity GABA site, but unable to form GABA channels. The requirement of a specific monomeric subunit (alpha 6 or gamma 2) for the tandem construct to express Cl--currents supports a pentameric structure of GABAA receptors consisting of two alpha 6, two beta 2, and one gamma 2 for the alpha 6 beta 2 gamma 2 and three alpha 6 and two beta 2 for the alpha 6 beta 2 subtype.
authors.affiliation: CNS Diseases Research, Upjohn Company, Kalamazoo, Michigan 49001, USA.
authors.forename: W B
authors.initials: WB
authors.lastname: Im
authors.forename: J F
authors.initials: JF
authors.lastname: Pregenzer
authors.forename: J A
authors.initials: JA
authors.lastname: Binder
authors.forename: G H
authors.initials: GH
authors.lastname: Dillon
authors.forename: G L
authors.initials: GL
authors.lastname: Alberts
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001569:Benzodiazepines / Q000378:metabolism; D001665:Binding Sites; D015152:Blotting, Northern; D002460:Cell Line; D018118:Chloride Channels / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D018076:DNA, Complementary; D015870:Gene Expression; D006801:Humans; D007668:Kidney; D007700:Kinetics; D046911:Macromolecular Substances; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009118:Muscimol / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D055503:Protein Multimerization; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000187:drug effects / Q000502:physiology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D000345:Affinity Labels; D001386:Azides; D018118:Chloride Channels; D018076:DNA, Complementary; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D001569:Benzodiazepines; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; C042957:Ro 15-4513