Abstract
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.
Footnotes
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This work was supported in part by National Institutes of Mental Health/National Research Service Award grant MH12966 (to J.A.T.) and National Institutes of Neurological Disorders and Stroke Grant NS34727 (to C.C.).
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A.M.K. and J.A.T. contributed equally to this work.
- Abbreviations:
- BZD
- benzodiazepine
- i-BZD
- imidazobenzodiazepine
- AChBP
- acetylcholine binding protein
- HEK
- human embryonic kidney
- MTS
- methanethiosulfonate
- MTSEA-Biotin
- N-biotinaminoethyl methanethiosulfonate
- MTSEA-Biotin-CAP
- N-biotincaproylaminoethyl methanethiosulfonate
- IGABA
- GABA-gated Cl−current
- Received August 7, 2002.
- Accepted October 22, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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