Residues at Positions 206 and 209 of the α1 Subunit of γ-Aminobutyric Acid_AReceptors Influence Affinities for Benzodiazepine Binding Site Ligands

Abstract

Ligands of the benzodiazepine binding site allosterically modulate gamma-aminobutyric acidA receptors. Their binding pocket is made up of amino acid residues located on both alpha and gamma subunits. We transiently expressed wild-type alpha1beta2gamma2 and mutant GABAA receptors in human embryonic kidney 293 cells and determined their binding properties. Receptors containing the mutant alphaY209A showed approximately 40-fold decrease in affinity for [3H]Ro 15-1788 and diazepam, whereas zolpidem displayed no measurable affinity. Receptors containing the mutant alphaY209F showed a small-to-moderate decrease in affinity for [3H]Ro 15-1788, diazepam, zolpidem, methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, and Cl 218872, amounting to 2-8-fold. Receptors containing the mutant alphaY209Q appeared in the surface membrane of transfected cells, bound [3H]muscimol with wild-type affinity, but failed to bind [3H]Ro 15-1788 or [3H]flunitrazepam with detectable affinity. If these mutant receptors were expressed in Xenopus laevis oocytes, the apparent affinity for GABA was only slightly decreased, whereas the ability of the currents to be stimulated by low concentrations of flunitrazepam was abolished. Receptors containing a point mutant of another amino acid residue, alphaT206A, surprisingly showed an increase in affinity of 5- and 16-fold, for the negative allosteric modulator methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and the partial positive allosteric modulator Cl 218872, respectively, whereas there was only a small decrease in affinity for Ro 15-1788, diazepam, and zolpidem, amounting to 2-, 4-, and 5-fold. Both alpha206 and alpha209 are thus both important in determining the binding affinities for ligands of the benzodiazepine binding site. The residues are spaced at an interval of three amino acids and may be part of an alpha helix.

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.…”

Structural Determinants for High-Affinity Zolpidem Binding to GABA-A receptors

“…4B), the pendant phenyl of flunitrazepam also participates in -stacking with ␣1His101, underscoring the importance of an aromatic residue at this position. Studies have shown that an aromatic ring at position 209 is crucial for maximal diazepam mediated modulation of GABA current as well as high-affinity zolpidem, flunitrazepam, and diazepam binding in ␣1␤2␥2 GABARs (Amin et al, 1997;Buhr et al, 1997b). In our dock, a potential hydrogen bond between ␣1Thr206 (loop C), and the nitro group (Fig.…”

The structural basis of function in Cys-loop receptors

“…For example, a1-His102 directly interacts with flunitrazepam and diazepam (Berezhnoy et al 2004 ;McKernan et al 1998 ;Tan et al 2007), while a1-Tyr160, a1-Tyr210 (Amin et al 1997) and c2-Phe77 (Buhr et al 1997a) form part of the aromatic-binding site for benzodiazepines. Residues a1-Thr206, a1-Glu209, a1-Tyr162, a1-Thr207, c2-Tyr58, c2-Ala79, c2-Met130 and c2-Thr142 contribute to benzodiazepine selectivity and efficacy Buhr et al 1997aBuhr et al , 1997bKucken et al 2000 ;Mihic et al 1994 ;Teissere & Czajkowski, 2001).…”