SUBTLE CHANGES IN RESIDUE-77 OF THE GAMMA-SUBUNIT OF ALPHA-1-BETA-2-GAMMA-2 GABA(A) RECEPTORS DRASTICALLY ALTER THE AFFINITY FOR LIGANDS OFTHE BENZODIAZEPINE BINDING-SITE

Recombinant alpha 1 beta 2 gamma 2 gamma-aminobutyric acid type A (GAB A(A)) receptors were functionally expressed in Xenopus oocytes, Upon t he mutation F77L, diazepam and Ro 15-1788 retained the ability to inte ract with the benzodiazepine binding site, but zolpidem lost this abil ity. To quantify these data, radioligand binding experiments were perf ormed using membrane preparations of transiently transfected human emb ryonic kidney 293 cells. The amino acid gamma 77, phenylalanine, was a lso mutated to tyrosine, tryptophan, and isoleucine. Although there wa s little effect on Ro 15-1788 binding upon mutation to tyrosine, the l oss in affinity for diazepam was from 12 to 2,720 nM. The change to le ucine, in contrast, resulted in little change in the diazepam affinity , whereas there was a strongly reduced affinity for zolpidem from 17 t o 4,870 nM and for methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carbo xylate (DMCM) from 1.9 to 1,780 nM, respectively. The change to trypto phan resulted in two-phasic displacement curves, and only about 50% of the [H-3]flunitrazepam binding could be displaced by zolpidem, DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam still res ulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in Xenopus o ocytes showed that all mutant channels displayed a comparatively small change (<4.3-fold) in their apparent agonist affinity and that these channels could still be functionally modulated by ligands of the benzo diazepine binding site. We conclude that subtle changes in gamma F77 d rastically affect benzodiazepine pharmacology and that this residue pr obably interacts directly with most ligands of the benzodiazepine bind ing site and therefore defines part of the benzodiazepine binding pock et.