Identification of amino acid residues responsible for the α5  subunit binding selectivity of L‐655,708, a benzodiazepine binding site ligand at the GABA_A receptor

Abstract

L-655,708 is a ligand for the benzodiazepine site of the g-aminobutyric acid type A (GABA A ) receptor that exhibits a 100-fold higher af®nity for a5-containing receptors compared with a1-containing receptors. Molecular biology approaches have been used to determine which residues in the a5 subunit are responsible for this selectivity. Two amino acids have been identi®ed, a5Thr208 and a5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher af®nity of this ligand at a5-containing receptors. L-655,708 is a partial inverse agonist at the GABA A receptor which exhibited no functional selectivity between a1-and a5-containing receptors and showed no change in ef®cacy at receptors containing a1 subunits where amino acids at both of the sites had been altered to their a5 counterparts (a1DSer205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also in¯uence the binding af®nities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA A receptor, and further delineate a region just N-terminal to the ®rst transmembrane domain of the receptor a subunit that contributes to this binding site. The mammalian g-aminobutyric acid type A (GABA A ) receptor is a pentameric structure containing different combinations of a1±6, b1±3, g1±3, d, 1 and u subunits (for reviews, see Barnard et al. 1998;Mehta and Ticku 1999). Among the many classes of drug which interact with the receptor are the benzodiazepines (BZs) that are used as anxiolytics, anticonvulsants and hypnotics. Immunoprecipitation with subunit speci®c antibodies, and recombinant receptor studies have demonstrated that high af®nity BZ-binding sites are found on receptors of abg 2 composition. The differential af®nity of drugs such as diazepam for such receptors in which the a1 subunit has been substituted by a6, demonstrating that the a subunit is a major contributor to the BZ binding site present on the receptor (Lu Èddens et al. 1990). Site-directed mutagenesis studies have been shown in a number of cases (e.g. Pritchett and Seeburg 1991;Wieland et al. 1992;Amin et al. 1997;Buhr et al. 1997) to identify residues within the a subunit which are critical for the drug±receptor interaction. Although the diazepam insensitivity of receptors containing a4 and a6 is well documented (e.g. Lu Èddens et al. 1990;Wisden et al. 1991), few clinically ef®cacious drugs other than zolpidem (which is 10-fold selective for a1-containing receptors over a2-and a3-containing receptors, with negligible af®nity for a4-, a5-and a6-containing receptors) discriminate between receptors containing other a subunits. Recently, however, the identi®cation of a ligand (L-655,708) with selectivity for the a5-containing receptor has been reported (Quirk et al.

Tonic inhibition in mouse hippocampal CA1 pyramidal neurons is mediated by α5 subunit-containing γ-aminobutyric acid type A receptors

“…2G Inset) and increased the charge transfer of the mIPSCs to 132.7 Ϯ 6.2% of control (n ϭ 15, P Ͻ 0.05). Binding studies showed that L-655,708 exhibits at least a 50-fold selectivity for ␣5␤2␥2 GABA A Rs compared with heteromeric complexes containing ␣1, ␣2, ␣3, or ␣6 subunits (31,32). Application of L-655,708 (5 M and 50 M; Fig.…”

Fluoxetine Increases GABAAReceptor Activity through a Novel Modulatory Site

“…To begin to elucidate the structures within the ␣ 5 subunit responsible for this property, we examined amino acid residues previously shown to be important in determining its pharmacological properties. The ␣ 5 subunit is also distinctive in its higher sensitivity to positive modulation by several benzodiazepine derivatives (Liu et al, 1996;Quirk et al, 1996;Strakhova et al, 2000), and an isoleucine residue (I215) in the extracellular domain was identified as important for this characteristic (Strakhova et al, 2000;Casula et al, 2001). This subunit also has a high sensitivity to inhibition by zinc, a property conferred by a unique histidine residue (H195) found only in the ␣ 5 subunit (Fisher, 2002b).…”

Multiple and Plastic Receptors Mediate Tonic GABAAReceptor Currents in the Hippocampus

“…A potential weakness of this argument is that the concentration of L-655,708 used in this study and the study by Caraiscos et al (2004) was deliberately chosen to be higher than the binding affinity for ␣5GABA A Rs (Casula et al, 2001) to maximize the sensitivity of this assay. However, it raises the possibility of nonspecific actions of L-655,708 [although 50 M L,655-708 had no detectable effect in ␣5GABA A R Ϫ/Ϫ mice (Caraiscos et al, 2004) and on the amplitude of sIPSCs in our experiments; p ϭ 0.12].…”