A tight coupling between β 2Y97 and β 2F200 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 (α 1R67, α 1R120, α 1R132, and β 2R207) and two aromatic residues (β 2Y97 and β 2F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α 1R67 nor α 1R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α 1R132 and both aromatic residues. Significant functional coupling between β 2R207 and both β 2Y97 and β 2F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β 2Y97 and β 2F200, and for the first time provided direct evidence for the involvement of β 2Y97 and β 2F200 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. © 2011 International Society for Neurochemistry.