Arg-274 and Leu-277 of the gamma-aminobutyric acid type A receptor alpha 2 subunit define agonist efficacy and potency.
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 gamma-aminobutyric acid type A receptor (GABA(A)-R). A conserved arginine residue in the TM2-TM3 loop of the GABA(A)-R alpha(2) subunit was mutated to alanine, and the mutant alpha(2)(R274A) was co-expressed with wild-type beta(1) and gamma(2S) subunits in human embryonic kidney (HEK) 293 cells. The GABA EC(50) was increased by about 27-fold in the mutant receptor relative to receptors containing a wild-type alpha(2) subunit. Similarly, the GABA EC(50) at alpha(2)(L277A)beta(1)gamma(2S) and alpha(2)(K279A)beta(1)gamma(2S) GABA(A)-R combinations was increased by 51- and 4-fold, respectively. The alpha(2)(R274A) or alpha(2)(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 alpha(2)(Arg-274) and alpha(2)(Leu-277) are crucial to the efficient transduction of agonist binding into channel gating at the GABA(A)-R.
O'Shea
S M
SM
Department of Neurobiology, University of Chicago, Chicago, Illinois 60637, USA.
Harrison
N L
NL
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D002460:Cell Line; D018755:GABA Agonists / Q000494:pharmacology*; D058785:GABA-A Receptor Agonists; D006801:Humans; D007930:Leucine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011963:Receptors, GABA-A / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018755:GABA Agonists; D058785:GABA-A Receptor Agonists; D011963:Receptors, GABA-A; D001120:Arginine; D007930:Leucine