Structural Elements of the γ-Aminobutyric Acid Type A Receptor Conferring Subtype Selectivity for Benzodiazepine Site Ligands

Abstract

␥-aminobutyric acid type A (GABA A ) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the ␣ 5 subunit were substituted by their corresponding ␣ 1 residues. Given the high affinity and selectivity of ␣ 1 -containing compared with ␣ 5 -containing GABA A receptors for zolpidem, mutated ␣ 5 subunits were co-expressed with ␤ 2 and ␥ 2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One ␣ 5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the ␣ 1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, ␣ 5 P162T and ␣ 5 E200G, might alter binding pocket conformation, whereas ␣ 5 T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the ␣ 1 subunit, particularly ␣ 1 -Ser 204, are the crucial residues influencing ligand selectivity at the binding pocket of ␣ 1 -containing receptors, and a model of this binding pocket is presented.

Structural Determinants for High-Affinity Zolpidem Binding to GABA-A receptors

“…5A), and potentially participate in longer range (4-7 Å) molecular interactions with zolpidem. It is noteworthy that ␣1Thr162 and ␣1Ser204 have previously been shown to confer binding selectivity for zolpidem to the ␣1 subunit (Renard et al, 1999). That our simulated molecular docking results are consistent with past experimental findings lends credence to the accuracy of our homology model and the prediction that flunitrazepam and zolpidem are situated differently within the BZD binding pocket.…”

“…␣1Ser204 in particular comes within 4 Å of zolpidem, making this residue a candidate for hydrogen bonding with the substrate. Replacing the corresponding residues in the zolpidem-insensitive ␣5 subunit with the aligned residues from the ␣1 subunit (i.e., ␣5P166T and ␣5T208S) significantly increased zolpidem affinity in ␣5␤2␥2 mutant receptors (Renard et al, 1999), further supporting our predictions.…”

“…Although site-directed mutagenesis and photoaffinity labeling studies have made significant strides in uncovering amino acid residues that contribute to the binding domains of classic BZDs, the residues that preferentially contribute to the binding of unconventional BZD-site ligands, such as zolpidem, remain unre-solved. A study exploiting the differences in zolpidem sensitivity between ␣1 and ␣5 subunit isoforms identified residues ␣1Thr162, ␣1Gly200, and ␣1Ser204 as contributing to the high zolpidem sensitivity of the ␣1 subunit isoform (Renard et al, 1999). Furthermore, replacing ␣1Gly200 with residues of increasing side-chain volume correlates with decreasing zolpidem affinity (Wingrove et al, 2002).…”

Structural Requirements for Imidazobenzodiazepine Binding to GABAA Receptors

“…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 Amin et al, 1997;Buhr et al, 1997b;Schaerer et al, 1998;Renard et al, 1999;Davies et al, 2001) and the ␥ subunit ( Buhr and Sigel, 1997;Buhr et al, 1997a;Wingrove et al, 1997;Kucken et al, 2000) that mediate high-affinity BZD binding; however, the specific interactions between individual amino acids and BZD ligands and the orientation of BZDs within the recognition site remain unclear (for review, see He et al, 2001). …”

Tracazolate Reveals a Novel Type of Allosteric Interaction with Recombinant γ-Aminobutyric AcidA Receptors

“…The loop C ligand-binding domain of the ␣ subunit has several amino acid positions that have been suggested to have a role in the function of benzodiazepine site ligands [e.g., positions 201 (Pritchett and Seeburg, 1991), 205 (Renard et al, 1999), and 207 and 210 (Amin et al, 1997;Buhr et al, 1997)]. Boileau et al (1998) and Boileau and Czajkowski (1999) have investigated the mechanism of benzodiazepine action using chimeric ␥2/␣1 subunits.…”