Despite the presence of the multiple subunits (␣, , ␥, and ␦) and their isoforms for ␥-aminobutyric acid, type A (GABA A ) receptors in mammalian brains, the ␣x2␥2 subtypes appear to be the prototype GABA A 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 [ 3 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 d (2.5 nM) and B max 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 A receptors consisting of two ␣6, two 2, and one ␥2 for the ␣62␥2 and three ␣6 and two 2 for the ␣62 subtype.GABA A 1 receptors, responsible for inhibitory neurotransmission in mammalian brains, are ligand-gated Cl Ϫ channels made of various subunits (␣, , ␥, and ␦) (1-3). Each subunit consists of several isoforms and contains four transmembranespanning segments (M1 to M4) (1-5). Despite the existence of the multiple subunits and their isoforms, combinations of ␣x, 2, and ␥2 subunits produced Cl Ϫ channels sharing many functional characteristics with native neuronal receptors and displaying the ability to respond to all the GABA A receptor ligands known up-to-date (1, 3, 6, 7). Such cloned GABA A receptors have been proposed to be of pentameric structure with M2 lining the pore in analogy with another member of the four transmembrane ligand-gated channel family, acetylcholine receptors (1-3). Recent studies, including immunoprecipitation with subunit specific antibodies, have shown the presence of two ␣ subunits per GABA A receptor (8 -11). Further experimental evidence is needed, however, about the stoichiometry of the recombinant GABA A receptors of ␣x2␥2 and their modes of association. One way to gain insight into this structural issue is to predetermine the alignment of subunits via gene fusion and to study such fused gene products. Similar approaches have been successful with potassium channels made of their subunits in concatameric or tandem linkages (12,13). In this study we prepared a tandem construct of ␣6 and 2 subunit cDNAs of th...