Despite the presence of the multiple subunits (, , , and ) and their isoforms for -aminobutyric acid, type A (GABA) receptors in mammalian brains, the x22 subtypes appear to be the prototype GABA 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 6 and 2 subunit cDNAs where the carboxyl-terminal of 6 is linked to the amino-terminal of 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 Cl currents, but its cotransfection with the cDNA for 6 or 2, but not 2, led to the appearance of GABA currents which were picrotoxin-sensitive and, in the case of 2 containing receptors, responded to a benzodiazepine agonist, U-92330. The high affinity GABA site, however, was detected with [H]muscimol binding in all combinations of the receptor subunits, including the tandem construct alone or with the 2. No appreciable differences were found in their K (2.5 nM) and B 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 (6 or 2) for the tandem construct to express Cl currents supports a pentameric structure of GABA receptors consisting of two 6, two 2, and one 2 for the 622 and three 6 and two 2 for the 62 subtype.

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