Identification of a residue in the γ‐aminobutyric acid type A receptor α subunit that differentially affects diazepam‐sensitive and ‐insensitive benzodiazepine site binding

Abstract

GABA A receptors that contain either the a4-or a6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including b-carbolines, bind to these diazepam-insensitive receptor subtypes. Some b-carbolines [e.g. ethyl b-carboline-3-carboxylate (b-CCE) and methyl 6,7-dimethoxy-4-ethyl-b-carboline-3-carboxylate (DMCM)] display a higher affinity for a4-compared to a6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric a6/a4 subunits and co-expressed these with wild-type rat b2 and c2 L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (a6N204/a4I203). Substitutions at a6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive a1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the b-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the a1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by 19-fold and 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the a subunit in diazepam-sensitive and -insensitive receptors. c-Aminobutyric acid type A (GABA A ) receptors are the major inhibitory receptors in the mammalian CNS. These receptors are members of the ligand-gated ion channel (LGIC) family that includes the nicotinic acetylcholine, glycine and serotonin type 3 (5-HT 3 ) receptors (Ortells and Lunt 1995). Each GABA A receptor is a heteropentamer of homologous subunits that assemble to form a GABA-gated, chloride-selective ion pore. Several different classes of receptor subunits have been cloned (a1-6, b1-3, c1-3, d, e, p and h) (for reviews, see Barnard et al. 1998;Korpi et al. 2002) and different combinations of these subunits in the oligomer determine the pharmacology of the receptor subtype. Although the number of subtypes remains unknown, most receptors appear to be formed by a, b and c subunits in a 2 : 2 : 1 stoichiometry (Tretter et al. 1997;Farrar et al. 1999). Allosteric modulation is a hallmark feature of the GABA A receptor and this is best exemplified by compounds that act at the benzodiazepine (BZD) binding site. Ligands of many chemical classes recognize this site and these compounds display a spectrum of efficacy from positive to negative allosteric modulation of GABA-gated chloride currents (Barnard et al. 1998).All receptors in the LGIC family share common structural features, including a similar subunit topology and overall receptor conformation. In all members of the family, agonist binding sites are thought to lie at subunit-subunit interfaces and to be formed by six distinct stretches or 'loop...

Selective targeting of the α5-subunit of GABAA receptors relaxes airway smooth muscle and inhibits cellular calcium handling

“…The ␣-subunit plays a critical role in ligand (both agonist and allosteric) binding. This is illustrated by the binding of classical 1,4 benzodiazepines at the interface of ␥-and ␣-subunits of ␣1-, ␣2-, ␣3-, and ␣5-subunit-containing GABA A receptors (43) but not at GABA A receptors containing ␣4-or ␣6-subunits (12,47). Furthermore, among the active benzodiazepine ␣-subunits (␣1, ␣2, ␣3, or ␣5), there are differential pharmacological profiles such that different ␣-subunits confer varied drug affinities.…”

A Conceptualization of Integrated Actions of Ethanol Contributing to its GABAmimetic Profile: A Commentary

“…Additionally, in vivo, the BZD-inverse agonists, RO15-4513 and FG7142, blocked the ethanol-induced depression of Purkinje neurons in the cerebellum (Palmer et al, 1988; see review by Palmer and Hoffer, 1990). The RO15-4513 and other BZD inverse agonists are now known to bind to receptors with a 6 -and a 4 -GABA A receptor subunits (Derry et al, 2004;Knoflach et al, 1996;Mhatre and Ticku, 1992;Yang et al, 1995), providing evidence that the reversal of ethanol action was related to their effect on these BZDinsensitive GABA A receptor subtypes.…”

“…Owing to the lack of sensitivity of BZD-insensitive receptors to zolpidem (Derry et al, 2004;Knoflach et al, 1996;Yang et al, 1995), these GABA A receptors containing the a 4 /a 6 /b X d subunits would not be viable candidates for ethanol action in brain regions with type-1-BZD (zolpidem) receptorsFsites where ethanol was shown earlier in vivo to support enhancement of GABA . Therefore, additional initiatives undertaken were directed at clarifying the means by which ethanol might display its influence on GABA transmission at sites lacking BZD-insensitive receptors by a mechanism distinct from a direct effect on GABA A receptor function.…”

Different Residues in the GABAA Receptor Benzodiazepine Binding Pocket Mediate Benzodiazepine Efficacy and Binding

“…Although multiple studies have identified residues that are involved in mediating the apparent binding affinity (K d ) of BZD-site ligands, including typical [1,4]benzodiazepines (Wieland and Lü ddens, 1994;Kucken et al, 2000;Boileau et al, 2002;Derry et al, 2004), cyclopyrrolones [e.g., eszopiclone (ESZ)] (Davies et al, 2000;, and imidazopyridines [e.g., zolpidem (ZPM)] (Buhr et al, 1996Schaerer et al, 1998;, much less is known about the structural determinants that couple their binding to modulation of I GABA and govern whether a BZD-site ligand is a positive modulator, zero modulator, or negative modulator (i.e., BZD efficacy).…”