Structural elements of the γ-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABA(A)) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha(5) subunit were substituted by their corresponding alpha(1) residues. Given the high affinity and selectivity of alpha(1)-containing compared with alpha(5)-containing GABA(A) receptors for zolpidem, mutated alpha(5) subunits were co-expressed with beta(2) and gamma(2) subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha(5) mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha(1) subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha(5)P162T and alpha(5)E200G, might alter binding pocket conformation, whereas alpha(5)T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem, These three amino acid substitutions also influenced receptor affinity for CL218872, Our data thus suggest that corresponding amino acids of the alpha(1) subunit, particularly alpha(1)-Ser(204), are the crucial residues influencing ligand selectivity at the binding pocket of alpha(1)-containing receptors, and a model of this binding pocket is presented.