Vol. 58, Issue 6, 1434-1440, December 2000

A Single Amino Acid Residue on the ₅ Subunit (Ile215) Is Essential for Ligand Selectivity at ₅₃₂ -Aminobutyric Acid_A Receptors

Marina I. Strakhova,¹ Scott C. Harvey, Christine M. Cook, James M. Cook, and Phil Skolnick

Neuroscience Discovery Research, Lilly Research Laboratories, Indianapolis, Indiana (M.I.S., S.C.H., C.M.C., P.S.); Department of Pharmacology and Toxicology, Indiana University School of Medicine, Indianapolis, Indiana (C.M.C., P.S.); and Department of Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin (J.M.C.)

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both high affinity and selectivity for GABA_A receptors containing an ₅ subunit. A single amino acid residue (₅Ile215) 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 ₅Ile215 with the cognate amino acid contained in the ₁ subunit (Val211) reduced the selectivity of RY-80 for ₅₃₂ receptors from ~135- to ~8-fold compared with ₁₃₂ receptors. This mutation produced a comparable reduction in the selectivity of RY-24 (a structural analog of RY-80) for ₅₃₂ receptors but did not markedly alter the affinities of ligands (e.g., flunitrazepam) that are not subtype-selective. Conversely, substitution of the ₁ subunit with the cognate amino acid contained in the ₅ subunit (i.e., ₁V211I) increased the affinities of ₅-selective ligands by a ~20-fold and reduced by 3-fold the affinity of an ₁-selective agonist (zolpidem). Increasing the lipophilicity (e.g., by substitution of Phe) of ₅215 did not significantly affect the affinities (and selectivities) of RY-80 and RY-24 for ₅-containing GABA_A 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 ₅I215V mutation. These data indicate that residue ₅215 may not participate in formation of the lipophilic L₂ pocket that has been proposed to contribute to the unique pharmacological properties of ₅-containing GABA_A receptors. RY-80 and RY-24 acted as inverse agonists in both wild-type ₅₃₂ and mutant ₅I215K₃₂ receptors expressed in Xenopus laevis oocytes. However, both RY-24 and RY-80 acted as antagonists at mutant ₅I215V₃₂ and ₅I215T₃₂ receptors, whereas the efficacy of flunitrazepam was similar at all three receptor isoforms. The data demonstrate that amino acid residue ₅215 is a determinant of both ligand affinity and efficacy at GABA_A receptors containing an ₅ subunit.