Arg-274 and Leu-277 of the gamma-aminobutyric acid type A receptor alpha 2 subunit define agonist efficacy and potency

J Biol Chem. 2000 Jul 28;275(30):22764-8. doi: 10.1074/jbc.M001299200.

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 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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Arginine / metabolism*
  • Cell Line
  • GABA Agonists / pharmacology*
  • GABA-A Receptor Agonists*
  • Humans
  • Leucine / metabolism*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Receptors, GABA-A / chemistry
  • Receptors, GABA-A / metabolism
  • Sequence Homology, Amino Acid

Substances

  • GABA Agonists
  • GABA-A Receptor Agonists
  • Receptors, GABA-A
  • Arginine
  • Leucine