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Volume 272, Number 18, Issue of May 2, 1997 pp. 11799-11804
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Subtle Changes in Residue 77 of the  Subunit of 122 GABA_A Receptors Drastically Alter the Affinity for Ligands of the Benzodiazepine Binding Site

(Received for publication, November 5, 1996, and in revised form, January 27, 1997)

From the Department of Pharmacology, University of Bern, CH-3010 Bern, Switzerland

Recombinant 122 -aminobutyric acid type A (GABA_A) receptors were functionally expressed in Xenopus oocytes. Upon the mutation F77L, diazepam and Ro 15-1788 retained the ability to interact with the benzodiazepine binding site, but zolpidem lost this ability. To quantify these data, radioligand binding experiments were performed using membrane preparations of transiently transfected human embryonic kidney 293 cells. The amino acid 77, phenylalanine, was also mutated to tyrosine, tryptophan, and isoleucine. Although there was little effect on Ro 15-1788 binding upon mutation to tyrosine, the loss in affinity for diazepam was from 12 to 2,720 nM. The change to leucine, in contrast, resulted in little change in the diazepam affinity, whereas there was a strongly reduced affinity for zolpidem from 17 to 4,870 nM and for methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate (DMCM) from 1.9 to 1,780 nM, respectively. The change to tryptophan resulted in two-phasic displacement curves, and only about 50% of the [³H]flunitrazepam binding could be displaced by zolpidem, DMCM, and Ro 15-1788, respectively, whereas midazolam and diazepam still resulted in 100% displacement, indicating the presence of two sites upon expression of this mutant receptor. Functional expression in Xenopus oocytes 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 benzodiazepine binding site. We conclude that subtle changes in F77 drastically affect benzodiazepine pharmacology and that this residue probably interacts directly with most ligands of the benzodiazepine binding site and therefore defines part of the benzodiazepine binding pocket.

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