Arg-274 and Leu-277 of the γ-aminobutyric acid type A receptor α2 subunit define agonist efficacy and potency

Abstract

Alanine-scanning mutagenesis and the whole cell voltage clamp technique were used to investigate the function of the extracellular loop between the second and third transmembrane domains (TM2-TM3) of the γ-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R α2 subunit was mutated to alanine, and the mutant α2(R274A) was co-expressed with wild-type β1 and γ(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC50 was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type α2 subunit. Similarly, the GABA EC50 at α2(L277A)-β1γ(2S) and α2(K279A)β1γ(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The α2(R274A) or α2(L277A) mutations also reduced the maximal response of piperidine-4-sulfonic acid relative to GABA by converting piperidine-4-sulfonic acid into a weak partial agonist at the GABA(A)-R. Based on these results, we propose that α2(Arg-274) and α2(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.