title: A tight coupling between β₂Y97 and β₂F200 of the GABA(A) receptor mediates GABA binding.
abstract: The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α₁R67, α₁R120, α₁R132, and β₂R207) and two aromatic residues (β₂Y97 and β₂F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α₁R67 nor α₁R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α₁R132 and both aromatic residues. Significant functional coupling between β₂R207 and both β₂Y97 and β₂F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β₂Y97 and β₂F200, and for the first time provided direct evidence for the involvement of β₂Y97 and β₂F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.
authors.affiliation: Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53201-1881, USA. phu.tran@marquette.edu
authors.forename: Phu N
authors.initials: PN
authors.lastname: Tran
authors.forename: Kurt T
authors.initials: KT
authors.lastname: Laha
authors.forename: David A
authors.initials: DA
authors.lastname: Wagner
mesh_terms: D000465:Algorithms; D001120:Arginine / Q000378:metabolism; D002478:Cells, Cultured; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D055724:Electrophysiological Phenomena; D018756:GABA Antagonists / Q000378:metabolism / Q000494:pharmacology; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D018076:DNA, Complementary; D018756:GABA Antagonists; C495710:GABRB2 protein, human; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D001120:Arginine