A tight coupling between β₂Y97 and β₂F200 of the GABA(A) receptor mediates GABA binding.

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.