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 (α&sub1;R67, α&sub1;R120, α&sub1;R132, and β&sub2;R207) and two aromatic residues (β&sub2;Y97 and β&sub2;F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α&sub1;R67 nor α&sub1;R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α&sub1;R132 and both aromatic residues. Significant functional coupling between β&sub2;R207 and both β&sub2;Y97 and β&sub2;F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β&sub2;Y97 and β&sub2;F200, and for the first time provided direct evidence for the involvement of β&sub2;Y97 and β&sub2;F200 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.