CHLORIDE CHANNEL EXPRESSION WITH THE TANDEM CONSTRUCT OF ALPHA-6-BETA-2 GABA(A) RECEPTOR SUBUNIT REQUIRES A MONOMERIC SUBUNIT OF ALPHA-6 ORGAMMA-2

Despite the presence of the multiple subunits (alpha, beta, gamma, and delta) and their isoforms for gamma-aminobutyric acid, type A (GABA(A )) receptors in mammalian brains, the alpha x beta 2 gamma 2 subtypes appear to be the prototype GABA(A) receptors sharing many properties w ith native neuronal receptors, In order to gain insight into their sub unit stoichiometry and orientation, we prepared a tandem construct of the alpha 6 and beta 2 subunit cDNAs where the carboxyl-terminal of al pha 6 is linked to the amino terminal of beta 2 via a linker encoding 10 glutamine residues. Transfection of human embryonic kidney 293 cell s with the tandem construct alone failed to induce GABA-dependent Cl- 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 pic rotoxin-sensitive and, in the case of gamma 2 containing receptors, re sponded to a benzodiazepine agonist, U-92330. The high affinity GABA s ite, however, was detected with [H-3]muscimol binding in all combinati ons of the receptor subunits, including the tandem construct alone or with the beta 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 consi stent with the view that the polypeptides arising from the tandem cons truct were expressed with the high affinity GABA site, but unable to f orm GABA channels, The requirement of a specific monomeric subunit (al pha 6 or gamma 2) for the tandem construct to express Cl- currents sup ports a pentameric structure of GABA(A) receptors consisting of two al pha 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.