A β-strand in the γ2 subunit lines the benzodiazepine binding site of the GABAA receptor:: Structural rearrangements detected during channel gating

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma (2)Y72-gamma (2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha (1) and beta (2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma (2)T73-gamma (2)T81 adopts a beta -strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma (2)A79 and gamma (2)T81, that line the BZD binding site. Sulfhydryl modification of gamma (2)A79C or gamma (2)T81C allosterically shifts the GABA EC50 of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.