ACCELERATION OF GABA-DEPENDENT DESENSITIZATION BY MUTATING THREONINE-266 TO ALANINE OF THE ALPHA-6 SUBUNIT OF RAT GABA(A) RECEPTORS

Various GABA(A) receptor subunits share four highly homologous putativ e transmembrane domains (M1 to M4) and have been proposed to form an i on channel of pentameric structure with M2 lining the pore. The carbox yl terminal side of M2 contains three amino acid residues containing a hydroxyl group, which are Thr 265, 266 and serine 268 in the alpha 6 subunit. In order to study their functional role, we generated mutants of the alpha 6 subunit carrying a single point mutation of threonine 265 or 266 to alanine, or serine 268 to glycine. Go-expression of the mutants with beta 2 and gamma 2 subunits in human embryonic kidney cel ls produced functional receptors which are similar to the wild type in their sensitivity to a benzodiazepine agonist (U-92330), insensitivit y to Zn, anion permeability, and GABA dose-response profiles as monito red with the whole cell patch clamp technique. Only in the alpha 6(T26 6A)beta 2 gamma 2 subtype, however, GABA-induced Cl- currents decayed much more rapidly than the wild type (about 10 times faster). Analysis of the GABA dependency of desensitization indicates that the T266A mu tation enhanced the desensitization rate with little effect on the rec overy rate from desensitization or on the half-maximal GABA concentrat ion. We conclude that threonine 266 in the alpha 6 subunit plays a piv otal role in desensitization processes of GABA(A) receptors.