Mechanism of α-subunit selectivity of benzodiazepine pharmacology at γ-aminobutyric acid type A receptors

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Abstract

Benzodiazepine pharmacology at the GABAA receptor is dependent on the α and γ subunit isoforms present. Ligands with higher affinity for certain isoforms—selective compounds—have been classified into benzodiazepine type I and II and into diazepam-sensitive and diazepam-insensitive receptors. A single amino acid position (α1G201/α3E225) has been identified which discriminates BZI and BZII receptors. The role of this residue has been explored by mutagenesis of α1 position 201 and the pharmacology of recombinant receptors examined using BZI receptor agonists. Ligand affinity is reduced by increasing side chain volume at α1G201 suggesting that steric inhibition underlies α-subunit selectivity. A second amino acid (α1H102/α6R100) determines diazepam sensitivity. The nature of the amino acid at this position was also examined by mutagenesis. Flumazenil and Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) binding affinity correlated weakly with the amino acid hydrophobicity suggesting a weak hydrophobic interaction between the ligand and α1H102.

Introduction

Benzodiazepines are a clinically important class of compounds used in the treatment of anxiety and sleep disorders. They exert their action by modulation of the GABAA receptor acting at a site distinct from that of the natural agonist, γ-aminobutyric acid (GABA). The GABAA receptor is a pentameric complex made up from a subset of six known subunit classes (α1–α6, β1–β3, γ1–γ3, δ, ε and θ; Barnard et al., 1998, Bonnert et al., 1999). The properties of receptors, including the benzodiazepine pharmacology, are dependent on the subunit composition with the most common receptor isoforms consisting of α, β and γ subunits (Whiting et al., 1995).

Benzodiazepine receptors have been classified pharmacologically into those which recognize the classical, 5-phenyl-1,4-benzodiazepine agonists (for example diazepam and flunitrazepam) referred to as ‘diazepam-sensitive’ and those which do not recognize these ligands referred to as ‘diazepam-insensitive’ (Malminiemi and Korpi, 1989). The cloning of cDNAs encoding subunit isoforms has allowed the expression of recombinant receptors of known composition and it is now understood that the diazepam-sensitive/diazepam-insensitive terms relate to the α subunit isoform contained within a typical αβγ2 receptor as differentiated by the displacement of [3H]Ro15-4513 (ethyl 8-azido-6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a]-[1,4]benzodiazepine-3-carboxylate) with diazepam. Hence diazepam-sensitive receptors contain α1, α2, α3 or α5 whereas diazepam-insensitive receptors contain either α4 or α6 (Lüddens et al., 1990). The former group can be further subdivided by sensitivity to CL218,872 (3-methyl-6-(3-[trifluoromethyl]phenyl)-1,2,4-triazolo[4,3-b]pyridazine) with the higher affinity α1-containing receptors referred to as BZI and the lower affinity BZII receptors containing α2, α3 or α5. Two amino acid positions have been identified which determine each of these α-subunit selective profiles.

The α subunits are predominantly labelled in photoaffinity labelling studies (for example, Stephenson et al., 1990). Peptide mapping of labelled subunits shows that [3H]flunitrazepam and [3H]Ro15-4513 become covalently attached to different domains of diazepam-sensitive receptors Davies et al., 1996, Davies and Dunn, 1998, Duncalfe et al., 1996, Duncalfe and Dunn, 1996. The site of photoincorporation of [3H]flunitrazepam was identified by micropeptide sequencing as histidine 102 (bovine α1 subunit numbering; Duncalfe et al., 1996). The presence of a histidine at position 101 of rat α1 (and equivalent position in other α subunits) had already been shown to determine the high affinity of the benzodiazepine agonist diazepam at α1-containing receptors over those containing α4 or α6 which have arginine at the homologous position 100 (Wieland et al., 1992).

The amino acid determining the α1 subunit selectivity of CL218,872 and of zolpidem has also been identified as α1G201 (Pritchett and Seeburg, 1991). At both positions 102 and 201, it is noticeable that the subunits conferring the higher affinity have the smaller amino acid suggesting a steric role for the α subunit in benzodiazepine selectivity. It may be speculated that a similar mechanism also underlies the greater than 1000-fold decrease in affinity for diazepam, flunitrazepam, zolpidem and CL218,872 at α6-containing receptors compared to those having α1 Hadingham et al., 1996, Lüddens et al., 1990.

To further test the hypothesis that the α subunit negatively affects affinity of benzodiazepine binding and to investigate the role of residues at positions 102 and 201, a number of amino acid substitutions of the α1 subunit were made by site-directed mutagenesis. The mutant α1 subunits were coexpressed transiently with β and γ2 subunit cDNAs in human embryonic kidney (HEK) 293 cells and the recombinant receptors characterized by radioligand binding assays to determine the affinities for selective and non-selective benzodiazepine site ligands. A functional analysis was also made for recombinant receptors expressed in Xenopus oocytes. The results suggest that steric and charge effects underlie the role of the α subunit in determining the selectivity and affinity of benzodiazepine site agonists. Results similar to parts of this study have been reported by Davies et al. (1998) and Dunn et al. (1999) as discussed below. These studies of the effect of mutations at rat α1H101 also conclude that the nature of the amino acid at this position influences ligand recognition and efficacy without finding any correlation with amino acid properties.

Section snippets

Site-directed mutagenesis

Cloning of human cDNAs encoding the α1, α3, α6, β1, β3 and γ2S subunits has been described previously Hadingham et al., 1993a, Hadingham et al., 1993b, Hadingham et al., 1996. Oligonucleotide-directed mutagenesis was performed as described previously incorporating a diagnostic restriction site (Wingrove et al., 1994). Mutants were identified by the presence of the diagnostic site and confirmed by DNA sequencing. The numbering of the residues discussed uses that appropriate for the species used

Radioligand binding data for receptors containing α1G201 mutant subunits

The α1 subunit was mutated to change α1G201 to alanine, aspartate, glutamate, methionine or valine and expressed in combination with cDNAs encoding β1 and γ2S. Saturation analysis was performed using the non-BZI/BZII receptor selective radioligand [3H]flumazenil (Table 1). Affinities (Ki) were determined by displacement of this binding for CL218,872 and zolpidem (Table 1). These compounds were chosen since their affinity for α3-containing receptors is known to be affected by mutation of α3E225G

Determination of BZI/BZII receptor pharmacology

The amino acid determining the α1 subunit selectivity of CL218,872 and of zolpidem has previously been identified (Pritchett and Seeburg, 1991). Here α1/α3 chimeric subunits and subsequently mutations of the α3 subunit were made and displacement of [3H]flumazenil by CL218,872 used to identify α1G201 as the determinant of high affinity binding. Substitution of the corresponding position (E225) of α3 with glycine resulted in increased affinity for CL218,872 and zolpidem. Hence, this position is

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