The modulatory action of loreclezole at the gamma-aminobutyric acid type A receptor is determined by a single amino acid in the beta 2 and beta 3 subunit.

Abstract

Type A yaminobutyric acid (GABAA) receptors of the mammalian nervous system are a family of ligandgated ion channels probably formed from the coassembly of different subunits (a1.6, Pl39 8) in the arrangement apy or ad8. The activation of these receptors by GABA can be modulated by a range ofcompounds acting at distinct allosteric sites. One such compound is the broad-spectrum anticonvulsant loreclezole, which we have recently shown to act via a specific modulatory site on the P subunit of the GABAA receptor. The action of loreclezole depends on the type of 13 subunit present in the receptor complex; receptors containing P2 or 13 subunits have >300-fold higher affinity for loreclezole than receptors containing a PI subunit. We have used this property to identify the amino acid residue in the P subunit that determines the subunit selectivity of loreclezole. Chimeric P1/12 human GABAA receptor subunits were constructed and coexpressed in Xenopus oocytes with human a, and y subunits. The chimera 131/P2Lys237-Gly334 conferred sensitivity to 1 IAM loreclezole. Within this region there are four amino acids that are conserved in P2 and 13 but differ in 1. By mutating single amino acids of the P1 subunit to the P2/13 equivalent, only the P1 mutation of Ser-290 -* Asn conferred potentiation by loreclezole. Similarly, mutation of the homologous residue in the 12 and P3 subunits to the Pi equivalent (Asn --Ser) resulted in loss of sensitivity to loreclezole. The affinity for GABA and the potentiation by flunitrazepam were unchanged in receptors coning the mutated 13 subunits. Thus, a single amino acid, 12 Asn-289 (P1 Asn-290), located at the carboxyl-terminal end of the putative channel-lining domain TM2, confers sensitivity to the modulatory effects of loreclezole.The mammalian type A )-aminobutyric acid (GABAA) receptor gene family is now known to consist of a number of subunit polypeptides (al-a6, (31-P3, 3v-y3. 6, pi-p2) (1, 2).There is an increased body of evidence that suggests that in neurons these subunits coassemble in the arrangement a(yor a(3S (3,4), probably as pentamers (by analogy with the related nicotinic receptor), to give a family of receptor subtypes that have different spatial and temporal patterns ofexpression (5).The activity of GABAA receptors can be allosterically modulated by a number of agents, including ethanol and neurosteroids, and the clinically important barbiturates and benzodiazepines (BZs) (6, 7). The BZ pharmacology of GABAA receptor subtypes has been studied in some detail.Only receptors made up of an a, 3, and 'y subunit exhibit high-affinity BZ binding (8), and the pharmacology is determined by the type of a (9-11) and y (12), but not the (, (13), subunit present, suggesting that this modulatory site is constituted by determinants on both the a and y subunits. The structural determinants that contribute to the other modulatory sites of the GABAA receptor are currently unclear.Loreclezole, {(Z)-1-[2-chloro-2-(2,4-dichlorophenyl) ethenyl]-1,2,4-triazole}, is a broad-spectrum anti...

Early developmental alterations in GABAergic protein expression in fragile X knockout mice

“…The selectivity of etomidate (as well as loreclezole) is dependent on an asparagine residue at position 265 of the GABA A receptor β2 subunit (Wingrove et al, 1994). The results from our sleep time study, together with the results from the motor activity analysis, lend credence the notion that a decrease in β2 protein expression translates to a functional loss of the GABA A receptor activity.…”

The interaction of the general anesthetic etomidate with the γ-aminobutyric acid type A receptor is influenced by a single amino acid

“…The results obtained with the chimeric ␤ 1-2 and ␤ 2-1 subunits demonstrate that structural elements that reside within the transmembrane domains, connecting loops, or carboxylterminal region of the ␤ subunit are important for both the GABA-modulatory and GABA-mimetic effects of etomidate. The GABA-modulatory effects of loreclezole exhibit a similar profile (18). For this anticonvulsant, site-directed mutagenesis studies have highlighted the importance of an asparagine residue (␤ 2 and ␤ 3 ) versus a serine residue (␤ 1 ) located toward the carboxyl-terminal end of the second transmembrane domain, the putative pore-forming region of the chloride ion channel (18).…”

“…of the ␤ subunit (an asparagine for ␤ 2 and ␤ 3 and a serine for ␤ 1 ) dictates the differential interaction of this anticonvulsant with the GABA A receptor (18). Similarly, for oocytes expressing ␣ 6 , ␥ 2L and a ␤ 1 subunit in which this serine was changed to an asparagine residue (␤ 1 S290N ), both the potency (EC 50 ϭ 1.6 Ϯ 0.3 M) and the maximal effect (E max ϭ 150 Ϯ 13%, n ϭ 5) of etomidate was increased relative to receptors (␣ 6 ␤ 1 ␥ 2L ) expressing the wild-type ␤ 1 subunit (Fig.…”

Salicylidene salicylhydrazide, a selective inhibitor ofβ1‐containing GABAAreceptors

“…Wingrove et al (1994) who expressed the b1/2 chimeras and some of the point mutations with a1 and g2s reported that the affinities for GABA were unchanged. In this study, the EC 20 values of most of the chimeras and point mutations were not significantly different from the wild-type receptors (the exceptions being a2b1g2s vs a2b2 1-237 b1 238-474 g2s and a2b1g2s vs a2b2N289Sg2s one-way ANOVA with Tukey Kramer post hoc test).…”