Two Tyrosine Residues on the α Subunit Are Crucial for Benzodiazepine Binding and Allosteric Modulation of γ-Aminobutyric Acid_AReceptors

Abstract

SUMMARYBenzodiazepines (BZs) exert their therapeutic effects in the mammalian central nervous system at least in part by modulating the activation of ␥-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 ␣1␤2␥2 GABA A receptors. Sitedirected mutagenesis was used to introduce point mutations into the ␣1 and ␥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 studied using two-electrode voltage-clamp and ligandbinding techniques. With this approach, we identified two tyrosine residues on the ␣1 subunit (Tyr159 and Tyr209) that when mutated to serine, dramatically impaired modulation by dzp. The Y209S substitution resulted in a Ͼ7-fold increase in the EC 50 for dzp, and the Y159S substitution nearly abolished dzp-mediated potentiation. Both of these mutations abolished binding of the high affinity BZ receptor antagonist [ 3 H]Ro 15-1788 to GABA A receptors expressed in HEK 293 cells. These tyrosine residues correspond to two tyrosines of the ␤2 subunit (Tyr157 and Tyr205) previously postulated to form part of the GABA-binding site. Mutation of the corresponding tyrosine residues on the ␥2 subunit produced only a slight increase in the EC 50 for dzp (ϳ2-fold) with no significant effect on the binding affinity of [3 H]Ro 15-1788. These data suggest that Tyr159 and Tyr209 of the ␣1 subunit may be components of the BZ-binding site on ␣1␤2␥2 GABA A receptors.BZs are frequently prescribed as anxiolytics, sedatives, anticonvulsants, and muscle relaxants (1-3). It is now generally accepted that these compounds exert their therapeutic effects, at least partly, by interacting with GABA A receptors in the brain (2-8). Thus, a substantial effort has been directed at understanding the molecular mechanism by which BZs modulate GABA A receptor function (9 -12).Molecular cloning studies (13-15) have revealed multiple classes and isoforms of GABA A receptor subunits in the mammalian brain (␣1-6, ␤1-4, ␥1-3, ␦). This diversity of ␣, ␤, and ␥ subunits allows the expression of a vast number of structurally unique GABA A receptor subtypes with distinct pharmacologies. Studies using exogenous expression, photoaffinity labeling, chimeric subunits, and site-directed mutagenesis have indicated that the ␣ subunit contributes a major component of the BZ-binding site and, depending on the subtype, can confer either BZ1 or BZ2 pharmacology on the GABA A receptor (16 -23). In particular, a histidine residue at position 101 (22) and a glycine residue at position 200 (21) have been implicated in BZ binding to the GABA receptor complex (Fig. 1).Although the ␣ subunit seems to form part of the BZbinding site, the presence of a ␥ subunit is essential for the normal modulatory actions of BZs on GABA A receptors (19,24,25; although see Ref. 26). The...

Forced Subunit Assembly in α1β2γ2 GABAAReceptors

“…The inferred arrangement of subunits around the channel pore is hypothetical, based on the findings that the GABAbinding site is located at intersubunit contacts between ␣ and ␤ subunits (17)(18)(19)(20)(21) and that homologous amino acid residues of ␣ and ␥ subunits form the benzodiazepine-binding pocket (22)(23)(24)(25)(26)(27)(28). The observation that assembly intermediates comprising ␣␥ or ␣␤ dimers displayed some benzodiazepine or agonist binding, respectively (29), supported conclusions drawn from the former mutation studies.…”

Maternal Behavior Regulates Benzodiazepine/GABAA Receptor Subunit Expression in Brain Regions Associated with Fear in BALB/c and C57BL/6 Mice

“…Point mutations have been identified within both subunits that are sufficient to eliminate BZ receptor binding (Amin et al, 1997;Wingrove et al, 1997), and animals bearing a null mutation of the g2 subunit show approximately an 85% loss of [ 3 H]flunitrazepam binding (Gunther et al, 1995).…”

Structural Elements of the γ-Aminobutyric Acid Type A Receptor Conferring Subtype Selectivity for Benzodiazepine Site Ligands

“…We can thus assume that this P162T mutation is responsible for a local reorganization of the zolpidem binding site, presumably allowing a better hydrophobic interaction with the pyridine heterocycle of zolpidem. Two tyrosines (␣ 1 -Tyr 159 and ␣ 1 -Tyr 161 ) closely situated to this proline have also been reported to be implicated in benzodiazepine binding (10,27), one affecting potentiation of GABA by benzodiazepines, the other affecting flumazenil affinity. These amino acids may interact with zolpidem, but, since they are present on ␣ 1 and ␣ 5 , substitution mutagenesis would not help to resolve this point.…”

“…Several other amino acids surround the BZ binding site. For instance, ␣ 1 Y159S and ␣ 1 Y209S substitutions affect diazepam-mediated potentiation of the GABA A receptor, and these mutants also abolish [ 3 H]Ro15-1788 binding to the receptor (10). The ␣ 1 Y161A, ␣ 1 T206A, and ␥ 2 F77A substitutions result in a 3-fold increase in potentiation by diazepam (11,12).…”