No abstract

Benzodiazepines (BZDs) exert their effects in the CNS by binding to a modulatory site on GABA(A) receptors. Individual amino acids have been implicated in BZD recognition and modulation of the GABA(A) receptor, but the secondary structure of the amino acids contributing to the BZD binding site has not been elucidated. In this report we used the substituted cysteine accessibility method to understand the structural dynamics of a region of the GABA(A) receptor implicated in BZD binding, gamma(2)Y72-gamma(2)Y83. Each residue within this region was mutated to cysteine and expressed with wild-type alpha(1) and beta(2) subunits in Xenopus oocytes. Methanethiosulfonate (MTS) reagents were used to modify covalently the engineered cysteines, and the subsequent effects on BZD modulation of the receptor were monitored functionally by two-electrode voltage clamp. We identified an alternating pattern of accessibility to sulfhydryl modification, indicating that the region gamma(2)T73-gamma(2)T81 adopts a beta-strand conformation. By monitoring the ability of BZD ligands to impede the covalent modification of accessible cysteines, we also identified two residues within this region, gamma(2)A79 and gamma(2)T81, that line the BZD binding site. Sulfhydryl modification of gamma(2)A79C or gamma(2)T81C allosterically shifts the GABA EC(50) of the receptor, suggesting that certain MTS compounds may act as tethered agonists at the BZD binding site. Last, we present structural evidence that a portion of the BZD binding site undergoes a conformational change in response to GABA binding and channel gating (opening and desensitization). These data represent an important step in understanding allosteric communication in ligand-gated ion channels.

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

The imidazopyridine zolpidem (Ambien)