PT - JOURNAL ARTICLE AU - Amy M. Kucken AU - Jeremy A. Teissére AU - James Seffinga-Clark AU - David A. Wagner AU - Cynthia Czajkowski TI - Structural Requirements for Imidazobenzodiazepine Binding to GABA<sub>A</sub> Receptors AID - 10.1124/mol.63.2.289 DP - 2003 Feb 01 TA - Molecular Pharmacology PG - 289--296 VI - 63 IP - 2 4099 - http://molpharm.aspetjournals.org/content/63/2/289.short 4100 - http://molpharm.aspetjournals.org/content/63/2/289.full SO - Mol Pharmacol2003 Feb 01; 63 AB - Several structural subclasses of ligands bind to the benzodiazepine (BZD) binding site of the GABAA receptor. Previous studies from this laboratory have suggested that imidazobenzodiazepines (i-BZDs, e.g., Ro 15-1788) require domains in the BZD binding site for high-affinity binding that are distinct from the requirements of classic BZDs (e.g., flunitrazepam). Here, we used systematic mutagenesis and the substituted cysteine accessibility method to map the recognition domain of i-BZDs near two residues implicated in BZD binding, γ2A79 and γ2T81. Both classic BZDs and i-BZDs protect cysteines substituted at γ2A79 and γ2T81 from covalent modification, suggesting that these ligands may occupy common volumetric spaces during binding. However, the binding of i-BZDs is more sensitive to mutations at γ2A79 than classic BZDs or BZDs that lack a 3′-imidazo substituent (e.g., midazolam). The effect that γ2A79 mutagenesis has on the binding affinities of a series of structurally rigid i-BZDs is related to the volume of the 3′-imidazo substituents. Furthermore, larger amino acid side chains introduced at γ2A79 cause correspondingly larger decreases in the binding affinities of i-BZDs with bulky 3′ substituents. These data are consistent with a model in which γ2A79 lines a subsite within the BZD binding pocket that accommodates the 3′ substituent of i-BZDs. In agreement with our experimental data, computer-assisted docking of Ro 15-4513 into a molecular model of the BZD binding site positions the 3′-imidazo substituent of Ro 15-4513 near γ2A79. The American Society for Pharmacology and Experimental Therapeutics