Mutagenesis of the rat alpha 1 subunit of the gamma-aminobutyric Acid(A) receptor reveals the importance of residue 101 in determining the allostericeffects 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 sh own that the residue occupying position 101 (rat numbering) of the alpha su bunit 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 alpha 1 subunit and c oexpressed the mutant subunits with beta 2 and gamma 2 subunits in Xenopus oocytes. The effects of flunitrazepam, Ro15-1788, and Ro15-4513 on GABA-gat ed currents were then examined using electrophysiological techniques. Three substitutions (F, Y, and Q) had little effect on the ability of flunitraze pam to potentiate GABA-induced currents and had relatively modest effects o n the EC50 value of the flunitrazepam response. Other mutations (K and E) r esulted in drastic reduction of flunitrazepam recognition. All substitution s also affected the EC50 values for Ro15-1788 and Ro15-4513, and some led t o dramatic changes in their efficacy. For example, H101Y, H101K, and H101Q produced receptors at which Ro15-1788 acted as a partial agonist (maximum p otentiation of 164, 159, and 130%, respectively), whereas Ro15-4513 acted a s a partial agonist at H101F, H101K, and H101E (potentiation of 122, 138, a nd 110%, respectively) and an antagonist at H101Y and H101Q. These results indicate that the characteristics of the residue at position 101 of the alp ha 1 subunit play a crucial role in determining the efficacy of benzodiazep ine-site ligands.