2 TYROSINE RESIDUES ON THE OF SUBUNIT ARE CRUCIAL FOR BENZODIAZEPINE BINDING AND ALLOSTERIC MODULATION OF GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS

Benzodiazepines (BZs) exert their therapeutic effects in the mammalian central nervous system at least in part by modulating the activation of gamma-aminobutyric acid (GABA)-activated chloride channels. To gain further insight into the mechanism of action of BZs on GABA receptors , we have been investigating structural determinants required for the actions of the BZ diazepam (dzp) on recombinant alpha 1 beta 2 gamma 2 GABA(A) receptors. Site-directed mutagenesis was used to introduce po int mutations into the alpha 1 and gamma 2 GABA(A) receptor subunits. Wild-type and mutant GABA(A) receptors were then expressed in Xenopus laevis oocytes or human embryonic kidney 293 (HEK 293) cells and studi ed using two-electrode voltage-clamp and ligand-binding techniques. Wi th this approach, we identified two tyrosine residues on the alpha 1 s ubunit (Tyr159 and Tyr209) that when mutated to serine, dramatically i mpaired modulation by dzp. The Y209S substitution resulted in a >7-fol d increase in the EC50 for dzp, and the Y159S substitution nearly abol ished dzp-mediated potentiation. Both of these mutations abolished bin ding of the high affinity BZ receptor antagonist [H-3]Ro 15-1788 to GA BA(A) receptors expressed in HEK 293 cells. These tyrosine residues co rrespond to two tyrosines of the beta 2 subunit (Tyr157 and Tyr205) pr eviously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on the gamma 2 subunit produced o nly a slight increase in the EC50 for dzp (similar to 2-fold) with no significant effect on the binding affinity of [H-3]Ro 15-1788. These d ata suggest that Tyr159 and Tyr209 of the alpha 1 subunit may be compo nents of the BZ-binding site on alpha 1 beta 2 gamma 2 GABA(A) recepto rs.