Mutagenesis of the rat alpha1 subunit of the gamma-aminobutyric acid(A) receptor reveals the importance of residue 101 in determining the allosteric effects of benzodiazepine site ligands.

The gamma-aminobutyric acid(A) (GABA(A)) receptor contains a binding site (or sites) for benzodiazepines and related ligands. Previous studies have shown that the residue occupying position 101 (rat numbering) of the alpha subunit is particularly important in determining how some of these compounds interact with the receptor. We have made multiple substitutions (F, Y, K, Q, and E) of the histidine at this position of the rat alpha1 subunit and coexpressed the mutant subunits with beta2 and gamma2 subunits in Xenopus oocytes. The effects of flunitrazepam, Ro15-1788, and Ro15-4513 on GABA-gated currents were then examined using electrophysiological techniques. Three substitutions (F, Y, and Q) had little effect on the ability of flunitrazepam to potentiate GABA-induced currents and had relatively modest effects on the EC(50) value of the flunitrazepam response. Other mutations (K and E) resulted in drastic reduction of flunitrazepam recognition. All substitutions also affected the EC(50) values for Ro15-1788 and Ro15-4513, and some led to dramatic changes in their efficacy. For example, H101Y, H101K, and H101Q produced receptors at which Ro15-1788 acted as a partial agonist (maximum potentiation of 164, 159, and 130%, respectively), whereas Ro15-4513 acted as a partial agonist at H101F, H101K, and H101E (potentiation of 122, 138, and 110%, respectively) and an antagonist at H101Y and H101Q. These results indicate that the characteristics of the residue at position 101 of the alpha1 subunit play a crucial role in determining the efficacy of benzodiazepine-site ligands.