Structural Requirements for Imidazobenzodiazepine Binding to GABA_A Receptors

Abstract

Several structural subclasses of ligands bind to the benzodiazepine (BZD) binding site of the GABA A 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, ␥ 2 A79 and ␥ 2 T81. Both classic BZDs and i-BZDs protect cysteines substituted at ␥ 2 A79 and ␥ 2 T81 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 ␥ 2 A79 than classic BZDs or BZDs that lack a 3Ј-imidazo substituent (e.g., midazolam). The effect that ␥ 2 A79 mutagenesis has on the binding affinities of a series of structurally rigid i-BZDs is related to the volume of the 3Ј-imidazo substituents. Furthermore, larger amino acid side chains introduced at ␥ 2 A79 cause correspondingly larger decreases in the binding affinities of i-BZDs with bulky 3Ј substituents. These data are consistent with a model in which ␥ 2 A79 lines a subsite within the BZD binding pocket that accommodates the 3Ј 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 3Ј-imidazo substituent of Ro 15-4513 near ␥ 2 A79.Benzodiazepines (BZDs) are therapeutic agents commonly used in the treatment of anxiety, sleeplessness, and epilepsy (Doble and Martin, 1996). BZDs exert their anxiolytic, hypnotic, and anticonvulsant effects by interacting with a unique modulatory site on the GABA A receptor, the main effector of neuronal inhibition within the central nervous system (Hevers and Lü ddens, 1998). The BZD binding site is on the extracellular surface of the GABA A receptor at an interface formed by the ␣ and ␥ subunits (Smith and Olsen, 1995;Sigel and Buhr, 1997). Several studies have identified residues on both the ␣ subunit (Duncalfe et al

Enhancement of GABAergic Activity: Neuropharmacological Effects of Benzodiazepines and Therapeutic Use in Anesthesiology

“…3). Imidazobenzodiazepines seem to possess structural requirements for binding that are distinct from classic 1,4-BZDs (Kucken et al, 2000(Kucken et al, , 2003.…”

Determinants of Zinc Potentiation on the α4 Subunit of Neuronal Nicotinic Receptors

“…A series of residues on the ␥2 subunit (Tyr58, Phe77, Ala79, and Thr81) have been identified as components of the benzodiazepine-binding site (Kucken et al, 2000(Kucken et al, , 2003Teissere and Czajkowski, 2001). Examination of aligned amino acid sequences (Le Novere and Changeux, 2001) and a model of the ␣1␤2␥2 GABA A receptor extracellular domain (Kucken et al, 2003) reveals that the benzodiazepine-binding site residue ␥2Thr81 (Teissere and Czajkowski, 2001) is in a location identical to that of ␣4Glu59. Although ␣4Glu59 may not directly coordinate zinc, it is close to the zinc potentiation site on neuronal nAChRs.…”

Alcohol- and Alcohol Antagonist-Sensitive Human GABAA Receptors: Tracking δ Subunit Incorporation into Functional Receptors

“…To find differences that might be responsible for lack of effects of classic BZs (such as DZ) on ␦ subunit-containing receptors, we explored regions of the ␦ subunit that are homologous to regions in the ␥ subunit that contribute to BZ binding pockets at ␣ϩ/␥Ϫ subunit interfaces (Kucken et al, 2003). Our attention was drawn to a histidine residue in the ␦ subunit (␦H68) that is an alanine residue (␥2A79) at the homologous position in the ␥2 subunit (see Fig.…”