A single amino acid residue on the alpha(5) subunit (Ile215) is essential for ligand selectivity at alpha(5)beta(3)gamma(2) gamma-aminobutyric acid(A) receptors

Imidazobenzodiazepines such as RY-80 have been reported to exhibit both hig h affinity and selectivity for GABA(A) receptors containing an alpha (5) su bunit. A single amino acid residue (alpha (5)Ile215) has been identified th at plays a critical role in the high-affinity, subtype-selective effects of RY-80 and structurally related ligands. Thus, substitution of alpha (5)Ile 215 with the cognate amino acid contained in the alpha (1) subunit (Val211) reduced the selectivity of RY-80 for alpha (5)beta (3)gamma (2) receptors from similar to 135- to similar to8-fold compared with alpha (1)beta (3)gam ma (2) receptors. This mutation produced a comparable reduction in the sele ctivity of RY-24 (a structural analog of RY-80) for alpha (5)beta (3)gamma (2) receptors but did not markedly alter the affinities of ligands (e.g., f lunitrazepam) that are not subtype-selective. Conversely, substitution of t he alpha (1) subunit with the cognate amino acid contained in the alpha (5) subunit (i.e., alpha (1)V211I) increased the affinities of alpha (5)-selec tive ligands by a similar to 20- fold and reduced by 3-fold the affinity of an alpha (1)-selective agonist (zolpidem). Increasing the lipophilicity (e .g., by substitution of Phe) of alpha (5)215 did not significantly affect t he affinities (and selectivities) of RY-80 and RY-24 for alpha (5)-containi ng GABA(A) receptors. However, the effect of introducing hydrophilic and or charged residues (e.g., Lys, Asp, Thr) at this position was no greater tha n that produced by the alpha (5)I215V mutation. These data indicate that re sidue alpha (5)215 may not participate in formation of the lipophilic L-2 p ocket that has been proposed to contribute to the unique pharmacological pr operties of alpha (5)-containing GABA(A) receptors. RY-80 and RY-24 acted a s inverse agonists in both wild-type alpha (5)beta (3)gamma (2) and mutant alpha (5)I215K beta (3)gamma (2) receptors expressed in Xenopus laevis oocy tes. However, both RY-24 and RY-80 acted as antagonists at mutant alpha (5) I215V beta (3)gamma (2) and alpha (5)I215T beta (3)gamma (2) receptors, whe reas the efficacy of flunitrazepam was similar at all three receptor isofor ms. The data demonstrate that amino acid residue alpha (5)215 is a determin ant of both ligand affinity and efficacy at GABA(A) receptors containing an alpha (5) subunit.