A Single Amino Acid Residue on the α5 Subunit (Ile215) Is Essential for Ligand Selectivity at α5β3γ2 γ-Aminobutyric AcidA Receptors

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABAA receptors containing an α5 subunit. A single amino acid residue (α5Ile215) has been identified that plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of α5Ile215 with the cognate amino acid contained in the α1 subunit (Val211) reduced the selectivity of RY-80 for α5β3γ2 receptors from ∼135- to ∼8-fold compared with α1β3γ2 receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for α5β3γ2 receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the α1subunit with the cognate amino acid contained in the α5subunit (i.e., α1V211I) increased the affinities of α5-selective ligands by a ∼20-fold and reduced by 3-fold the affinity of an α1-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of α5215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for α5-containing GABAA receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater than that produced by the α5I215V mutation. These data indicate that residue α5215 may not participate in formation of the lipophilic L2 pocket that has been proposed to contribute to the unique pharmacological properties of α5-containing GABAA receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type α5β3γ2 and mutant α5I215Kβ3γ2 receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant α5I215Vβ3γ2 and α5I215Tβ3γ2 receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue α5215 is a determinant of both ligand affinity and efficacy at GABAA receptors containing an α5subunit.