Four amino acid exchanges convert a diazepam-insensitive, inverse agonist-preferring GABAA receptor into a diazepam-preferring GABAA receptor.
Benzodiazepines (BZ) exert their effects through GABAA receptors, which belong to the superfamily of ligand-gated ion channels. Coexpression of recombinant alpha, beta, and gamma subunits in a cell culture system mimics the BZ binding sites. The alpha variants largely determine the nature of the BZ binding site in such alpha i beta j gamma k heteromultimers (i = 1-6; j = 1-3; k = 1-3). Notably, the alpha 1 and alpha 6 variants confer high and low affinity for BZ agonists to the resulting receptor subtype, respectively. Glycine/glutamate and histidine/arginine positions in the alpha subunits of alpha x beta 2 gamma 2 receptors are involved in BZ I versus BZ II type selectivity. We now identify four amino acids in alpha 6 which together increase the affinity of the mutant alpha x beta 2 gamma 2 receptor for classical BZ receptor agonists above the level seen for any wild-type GABAA/BZ receptor. The most pronounced effect was due to an isoleucine to valine exchange. It simultaneously decreased the affinity for the BZ partial inverse agonist Ro 15-4513 20-fold and increased the affinity for diazepam 4-fold. The four amino acid residues stretch over most part of the N-terminal extracellular domain of the alpha subunit, suggesting that amino acids distant in the primary sequence form the BZ binding pocket.
Wieland
H A
HA
Laboratory for Molecular Neuroendocrinology, Center for Molecular Biology, Heidelberg, Germany.
Lüddens
H
H
D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001483:Base Sequence; D001569:Benzodiazepines / Q000378:metabolism; D001665:Binding Sites; D003975:Diazepam / Q000378:metabolism*; D018755:GABA Agonists / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011963:Receptors, GABA-A / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001386:Azides; D018755:GABA Agonists; D011963:Receptors, GABA-A; D001569:Benzodiazepines; C042957:Ro 15-4513; D003975:Diazepam