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...

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Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N‐methyl‐d‐aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C‐terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N‐terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C‐terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC50 values of 2.14 and 2.05 μM for glutamate and glycine, respectively, and an IC50 of 46.8 μM for Mg2+. Responses were antagonised by d‐2‐amino‐5‐phosphonovalerate, L‐689,560, pH 6.3, zinc, and MK‐801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.