High Level Expression of the NMDAR1 Glutamate Receptor Subunit in Electroporated COS Cells

Abstract: The rat N -methyl- d -aspartate (NMDA) glutamate receptor subunit NR1-1a was transiently expressed in COS cells using the technique of electroporation, which was fivefold more efficient than the calcium phosphate precipitation method of transfection. The glycine site antagonist 5,7-[³H]dichlorokynurenic acid labeled a single high-affinity site ( K _D = 29.6 ± 6 n M ; B _max = 19.4 ± 1.6 pmol/mg of protein) in membranes derived from COS cells electroporated with NR1-1a. In contrast to previous reports using transiently transfected human embryonic kidney 293 cells, binding of the noncompetitive antagonist (+)-5-[³H]methyl-10,11-dihydro-5 H -dibenzo[ a,d ]-cyclohepten-5,10-imine ([³H]MK-801) was not detected in NR1-1a-transfected COS cells. Although immunofluorescent labeling of electroporated COS cells demonstrated that the NR1-1a protein appears to be associated with the cell membrane, neither NMDA nor glutamate effected an increase in intracellular calcium concentration in fura-2-loaded cells, suggesting that homomeric NR1-1a receptors do not act as functional ligand-gated ion channels. Therefore, COS cells appear to differ from Xenopus oocytes with respect to the transient expression of functional homomeric NR1 receptors. Although expression of NR1-1a is sufficient to reconstitute a glycine binding site with wild-type affinity for antagonists in COS cells, recombinant homomeric NR1-1a receptors do not display properties that are characteristic of native NMDA receptors, such as permeability to Ca²⁺ and channel occupancy by MK-801, when expressed in this mammalian cell line.     

Principal role of NR3 subunits in NR1/NR3 excitatory glycine receptor function

Calcium-permeable N-methyl-d-aspartate (NMDA) receptors are tetrameric cation channels composed of glycine-binding NR1 and glutamate-binding NR2 subunits, which require binding of both glutamate and glycine for efficient channel gating. In contrast, receptors assembled from NR1 and NR3 subunits function as calcium-impermeable excitatory glycine receptors that respond to agonist application only with low efficacy. Here, we show that antagonists of and substitutions within the glycine-binding site of NR1 potentiate NR1/NR3 receptor function up to 25-fold, but inhibition or mutation of the NR3 glycine binding site reduces or abolishes receptor activation. Thus, glycine bound to the NR1 subunit causes auto-inhibition of NR1/NR3 receptors whereas glycine binding to the NR3 subunits is required for opening of the ion channel. Our results establish differential roles of the high-affinity NR3 and low-affinity NR1 glycine-binding sites in excitatory glycine receptor function.     

Developmental Changes of Cerebral Cortical [3H]Clonidine Binding in Rats: Influences of Guanine Nucleotide and Cations

Abstract: Cerebral cortical [³H]clonidine binding and influences of GTP and cations were investigated in developing rats. The results from Scatchard plots were compatible with the presence of two populations of binding sites [high-affinity binding ( K_D = 0.59 n M ) and low-affinity binding ( K_D = 7.12 n M )] in 70-day-old rats but only high-affinity binding ( K_D = 0.27 n M ) on day 1. Low-affinity binding was detectable on day 7. K_D values in high- and low-affinity binding were not significantly changed during development after 7 days. B_max of high-affinity binding reached a peak on day 15, and the value of low-affinity binding gradually increased with age. The addition of 10 μ M GTP caused a significant reduction in B_max of high-affinity binding after day 7. Neither K _D nor B_max of low-affinity binding was affected by 10 μ M GTP during development. NaCl (10 and 100 m M ) diminished the binding on days 7 and 70. MnCl₂ (0.1 and 1.0 m M ) markedly increased the binding on days 15 and 70 but not on day 7. It is suggested that: (1) single binding sites of α₂-adrenoceptors with higher affinity seem to be present on day 1; (2) low-affinity binding appears on day 7; (3) the number of high-affinity binding sites reaches a peak on day 15, followed by changes in populations of high-affinity as well as low-affinity sites without changing affinity; (4) the regulatory mechanism in α₂-receptors by guanine nucleotide reaches functional maturity between days 1 and 7; and (5) the involvement of Na⁺ and Mn²⁺ in α₂-receptor binding becomes functional by days 7 and 15, respectively.     

Characterization, Distribution, and Protein Kinase C-Mediated Regulation of [35S]Glutathione Binding Sites in Mouse and Human Spinal Cord

Abstract: We have characterized a high-affinity [³⁵S]-glutathione ([³⁵S]GSH) binding site in mouse and human spinal cord. [³⁵S]GSH binding sites in mouse and human spinal cord were observed largely within the gray matter in both the dorsal and ventral horns of spinal cord at cervical, thoracic, and lumbosacral segments. High-affinity [³⁵S]GSH binding was saturable, showing a B _max of 72 fmol/mg of protein and a K _D of 3.0 n M for mouse spinal cord and a B _max of 52 fmol/mg of protein and a K _D of 1.6 n M for human spinal cord. [³⁵S]GSH binding was displaceable by GSH, l -cysteine, and S -hexyl-GSH, but not by glutamate, glycine, or NMDA. These [³⁵S]GSH binding sites exhibited kinetic and saturation characteristics similar to GSH binding sites in rat brain astrocytes. To determine whether [³⁵S]GSH binding sites could be regulated by protein kinase C, we exposed human spinal cord sections to phorbol 12,13-diacetate for 1 h before ligand binding. Phorbol ester treatment increased [³⁵S]GSH binding by ∼60%, an effect that could be blocked by exposure of spinal cord sections to 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a general protein kinase inhibitor. [³⁵S]GSH binding sites in the spinal cord of both species exhibited many of the characteristics of a receptor including saturable binding, high affinity, ligand specificity, and modulation by kinase activity. These data suggest that GSH is a neurotransmitter in the CNS.     

Comparison of Solubilised Kainate and α-Amino-3-Hydroxy-5-Methylisoxazolepropionate Binding Sites in Chick Cerebellum

Abstract: [³H]Kainate bound to chick cerebellar membranes with a K _D of 0.6 μ M and with an exceptionally high B max of 165 pmol/mg of protein. In octylglucoside-solubilised extracts, the affinity of [³H]kainate was reduced ( K _D= 2.7 μ M ), but the B _max was relatively unchanged (130 pmol/mg of protein). The rank potency of competitive ligands was domoate > kainate > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > glutamate. Binding sites for α-[³H]amino-3-hydroxy-5-methylisoxazolepropionate ([³H]AMPA) were much less abundant, with K _D and B _max values in membranes of 86 n M and I pmol/mg of protein, respectively. The affinity of [³H]AMPA binding was also reduced on solubilisation ( K _D= 465 n M ), but there was an increase in the B _max (1.7 pmol/mg of protein). Quisqualate and CNQX were the most effective displacers of [³H]AMPA binding, but kainate was also a relatively potent inhibitor. However, in contrast to the displacement profile for [³H]kainate, domoate was markedly less potent than kainate at displacing [³H]AMPA. These results suggest that [³H]AMPA binds to a small subset of the kainate sites that, unlike the majority of the [³H]kainate binding protein, which has been reported to be located in the Bergmann glia, may represent neuronal unitary non- N -methyl-D-aspartate receptors.     

Molecular Cloning, Expression, and Pharmacological Characterization of humEAA1, a Human Kainate Receptor Subunit

Abstract: Kainate is a potent neuroexcitatory agent; its neurotoxicity is thought to be mediated by an ionotropic receptor with a nanomolar affinity for kainate. In this report, we describe the cloning of a cDNA encoding a human glutamate ionotropic receptor subunit protein from a human hippocampal library. This cDNA, termed humEAA1, is most closely related to rat and human cDNAs for kainate receptor proteins and, when expressed in COS or Chinese hamster ovary cells, is associated with high-affinity kainate receptor binding. We have successfully established cell lines stably expressing humEAA1. This is the first report of establishment of stable cell lines expressing a glutamate receptor subunit. The relative potency of compounds for displacing [³H] kainate binding of humEAA1 receptors expressed in these stable cell lines was kainate > quisqualate > domoate > L-glutamate > ( RS )-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid > dihydro-kainate > 6, 7-dinitroquinoxaline-2, 3-dione > 6-cyano-7-nitroquinoxaline-2, 3-dione. Homooligomeric expression of humEAA1 does not appear to elicit ligand-gated ion channel activity. Nevertheless, the molecular structure and pharmacological characterization of high-affinity kainate binding of the humEAA1 expressed in the stable cell line (ppEAA1–16) suggest that the humEAA1 is a subunit protein of a human kainate receptor complex.     

Generation and Characterisation of Stable Cell Lines Expressing Recombinant Human N-Methyl-d-Aspartate Receptor Subtypes

Abstract: Transfection of mouse L(tk-) cells with human N -methyl- d -aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP 39653) had high affinity for NR1a/NR2A receptors ( K _D = 3.93 n M ) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6–5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.     

Characterization of 5,7-Dichlorokynurenate-Insensitive d-[3H]Serine Binding to Synaptosomal Fraction Isolated from Rat Brain Tissues

Abstract: To explore target sites for endogenous d -serine that are different from the glycine site of the N -methyl- d -aspartate (NMDA) type glutamate receptor, we have studied the binding of d -[³H]serine to the synaptosomal P2 fraction prepared from the rat brain and peripheral tissues in the presence of an excess concentration (100 µ M ) of the glycine site antagonist 5,7-dichlorokynurenate (DCK). Nonspecific binding was defined in the presence of 1 m M unlabeled d -serine. Association, dissociation, and saturation experiments indicated that d -[³H]serine bound rapidly and reversibly to a single population of recognition sites in the cerebellar P2 fraction in the presence of DCK, with a K _D of 614 n M and a B _max of 2.07 pmol/mg of protein. d -Serine, l -serine, and glycine produced a total inhibition of the specific DCK-insensitive d -[³H]serine binding to the cerebellum with similar K _i values. Strychnine and 7-chlorokynurenate failed to inhibit the binding at 10 µ M . The profiles of displacement of the DCK-insensitive d -[³H]serine binding by various amino acids and glutamate and glycine receptor-related compounds differ from those of any other defined recognition sites. DCK-insensitive d -[³H]serine binding was at high levels in the cerebral cortex and cerebellum but very low in the kidney and liver. The present findings indicate that the DCK-insensitive d -[³H]serine binding site could be a novel candidate for a target for endogenous d -serine in mammalian brains.     

Structural model of the N-methyl-D-aspartate receptor glycine site probed by site-directed chemical coupling

The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.     

NMDA Receptors in Rat Cerebellum and Forebrain: Subtle Differences in Pharmacology and Modulation

Abstract: Binding of [³H]glutamate, [³H]glycine, and the glutamate antagonist [³H]CGS-19755 to NMDA-type glutamate receptors was examined in homogenates of rat forebrain and cerebellum. Most glutamate agonists had a higher affinity at the [³H]glutamate binding site of cerebellar NMDA receptors as compared with forebrain, whereas all the glutamate antagonists examined showed the reverse relationship. The [³H]glycine binding site of forebrain and cerebellar NMDA receptors showed a similar pharmacology in both brain regions. In the cerebellum, however, [³H]glycine bound to a second site with a 10-fold lower affinity and with a pharmacology that resembled that of the glycine/strychnine chloride channel. [³H]Glutamate binding was not affected by glycine agonists or antagonists, nor was [³H]glycine binding affected by glutamate agonists in either forebrain or cerebellum. Both CGS-19755 and 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, glutamate antagonists, reduced [³H]glycine binding in cerebellum, whereas only CGS-19755 was effective in forebrain. Glycine agonists and antagonists modulated [³H]CGS-19755 binding in forebrain and cerebellum to different extents in the two brain regions. From these studies we conclude that the cerebellar NMDA receptor has a different pattern of modulation at glutamate and glycine sites and that glycine may play a more important role in the control of NMDA function in the cerebellum as compared with forebrain.     

[3H]Dizocilpine Association Kinetics Distinguish Stimulatory and Inhibitory Polyamine Sites of N-Methyl-d-Aspartate Receptors

Abstract: Spermine and other polyamines both stimulate and inhibit N -methyl- d -aspartate receptor function, probably by interacting with two separate sites. To characterize these two actions, the effect of spermine on the binding kinetics of the channel blocker [³H]dizocilpine was studied in the presence of glutamate and glycine. Low concentrations (10 µ M ) of spermine increased the association and dissociation rates without modifying equilibrium binding, indicating that spermine increases the accessibility of [³H]dizocilpine to the channel by interacting with a high-affinity, stimulatory site. At higher concentrations (1 m M ), spermine markedly decreased equilibrium [³H]-dizocilpine binding by decreasing both affinity and B _max, indicating that spermine allosterically inhibits binding by interacting with a second, low-affinity site. The presumed polyamine antagonists arcaine, diethylenetriamine, and 1,10-diaminodecane completely inhibited equilibrium [³H]dizocilpine binding, probably by interacting with the inhibitory polyamine site or other sites, but not with the stimulatory polyamine site. Low concentrations (10 µ M ) of ifenprodil completely reversed the increase in association rate produced by spermine, whereas higher concentrations (IC₅₀ = 123 µ M ) inhibited equilibrium binding, indicating that ifenprodil is both a potent antagonist of the stimulatory site and a low-affinity ligand of the inhibitory site. The polyamine agonists spermine, spermidine, and neomycin interacted with the inhibitory site, but produced only partial inhibition of equilibrium [³H]dizocilpine binding.     

Allosteric Modulation of [3H]CGP 39653 Binding by Glycine in Rat Brain

Abstract: D,L-(E)-2-Amino-4-propyl-5-phosphono-3-pen-tenoic acid (CGP 39653). a new, high-affinity, selective NMDA receptor antagonist, interacts with rat cortical membranes in a saturable way and apparently to a single binding site, with a K_D of 10.7 nM and a receptor density of 2.6 pmol/mg of protein. Displacement analysis of [³H]CGP 39653 binding shows a pharmacological profile similar to that reported for another NMDA antagonist, 3-[(±)-2-carboxypiperazin-4-yI]propyl-1-phosphonic acid (CPP). Glycine, however, is able to discriminate between the two ligands; in fact, it does not affect [³H]CPP binding but inhibits [³H]CGP 39653 binding in a biphasic way. D-Serine, another agonist at the strychnine-insensitive glycine binding site of the NMDA receptor complex, inhibits [³H]CGP 39653 binding in the same way as glycine, with a potency that correlates with its binding affinity at the glycine site. In addition, 7-chlorokynurenic acid, an antagonist at the glycine site, is able to reverse the displacement of [³H]CGP 39653 by glycine in a dose-dependent manner. Furthermore, the dissociation rate constant of [³H]CGP 39653 is enhanced in the presence of glycine, whereas the presence of NMDA receptor ligands does not modify the rate of dissociation of [³H]CGP 39653 from the receptor. These results indicate that part of the binding of the NMDA antagonist CGP 39653 can be potently modified by glycine through an allosteric mechanism, and suggest the existence of two antagonist preferring NMDA receptor subtypes that are differentially modulated through the glycine binding site.     

Tyrosine hydroxylase activity is regulated by two distinct dopamine-binding sites

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline, is regulated acutely by feedback inhibition by the catecholamines and relief of this inhibition by phosphorylation of serine 40 (Ser40). Phosphorylation of serine 40 abolishes the binding of dopamine to a high affinity ( K _D < 4 nM) site on TH, thereby increasing the activity of the enzyme. We have found that TH also contains a second low affinity ( K _D = 90 nM) dopamine-binding site, which is present in both the non-phosphorylated and the Ser40-phosphorylated forms of the enzyme. Binding of dopamine to the high-affinity site decreases V _max and increases the K _m for the cofactor tetrahydrobiopterin, while binding of dopamine to the low-affinity site regulates TH activity by increasing the K _m for tetrahydrobiopterin. Kinetic analysis indicates that both sites are present in each of the four human TH isoforms. Dissociation of dopamine from the low-affinity site increases TH activity 12-fold for the non-phosphorylated enzyme and 9-fold for the Ser40-phosphorylated enzyme. The low-affinity dopamine-binding site has the potential to be the primary mechanism responsible for the regulation of catecholamine synthesis under most conditions.     

Neuroprotective Effects of NMDA Receptor Glycine Recognition Site Antagonism: Dependence on Glycine Concentration

Abstract: High-affinity NMDA receptor glycine recognition site antagonists protect brain tissue from ischemic damage. The neuroprotective effect of 5-nitro-6,7-dichloro-2,3-quinoxalinedione (ACEA 1021), a selective NMDA receptor antagonist with nanomolar affinity for the glycine binding site, was examined in rat cortical mixed neuronal/glial cultures. ACEA 1021 alone did not alter spontaneous lactate dehydrogenase (LDH) release. Treatment with ACEA 1021 (0.1–10 µ M ) before 500 µ M glutamate, 30 µ M NMDA, or 300 µ M kainate exposure was found to reduce LDH release in a concentration-dependent fashion. These effects were altered by adding glycine to the medium. Glycine (1 m M ) partially reversed the effect of ACEA 1021 on kainate cytotoxicity. Glycine (100 µ M –1 m M ) completely blocked the effects of ACEA 1021 on glutamate and NMDA cytotoxicity. The glycine concentration that produced a half-maximal potentiation of excitotoxin-induced LDH release in the presence of 1.0 µ M ACEA 1021 was similar for glutamate and NMDA (18 ± 3 and 29 ± 9 µ M , respectively). ACEA 1021 also reduced kainate toxicity in cultures treated with MK-801. The effects of glycine and ACEA 1021 on glutamate-induced LDH release were consistent with a model of simple competitive interaction for the strychnine-insensitive NMDA receptor glycine recognition site, although nonspecific effects at the kainate receptor may be of lesser importance.     

Characterization of Glutamate Binding Sites in Receptors Assembled from Transfected NMDA Receptor Subunits

Abstract: Previous studies in brain and recombinant NMDA receptors have observed heterogeneity in NMDA-sensitive glutamate binding site. We further characterized the glutamate site assembled from NR1a, NR2A, and NR2B NMDA receptor subunits using l -[³H]glutamate and [³H]CGP 39653 binding assays. In contrast to earlier reports, we demonstrate a unique pharmacology for the NR2A subunit alone, which has high affinity for agonists but low affinity for competitive antagonists compared with heteromeric combinations of NR1a + NR2A and NR1a + NR2B. Similar to previous reports, we find unequal antagonist affinity between heteromeric combinations of NR1a + NR2A and NR1a + NR2B. However, unlike earlier reports, we describe two binding components within each heteromeric transfection that more closely resemble data obtained for binding to brain membranes. In addition, we show Mg²⁺ can alter [³H]CGP 39653 binding in both the NR1a + NR2A and the NR1a + NR2B combination, thus allowing comparison of the [³H]CGP 39653-labeled site between the two heteromeric combinations. Agonist inhibition of [³H]CGP 39653 binding revealed differences between the heteromeric combinations as well as within each heteromeric combination, the latter of which more closely resembled results from brain. These results further determine components of the agonist and antagonist binding sites of the NMDA receptor as well as suggest additional possible mechanisms of heterogeneity of the glutamate site in the brain.     

Thiamine Deficiency in Cultured Neuroblastoma Cells: Effect on Mitochondrial Function and Peripheral Benzodiazepine Receptors

Abstract: When neuroblastoma cells were transferred to a medium of low (6 n M ) thiamine concentration, a 16-fold decrease in total intracellular thiamine content occurred within 8 days. Respiration and ATP levels were only slightly affected, but addition of a thiamine transport inhibitor (amprolium) decreased ATP content and increased lactate production. Oxygen consumption became low and insensitive to oligomycin and uncouplers. At least 25% of mitochondria were swollen and electron translucent. Cell mortality increased to 75% within 5 days. [³H]PK 11195, a specific ligand of peripheral benzodiazepine receptors (located in the outer mitochondrial membrane) binds to the cells with high affinity ( K _D = 1.4 ± 0.2 n M ). Thiamine deficiency leads to an increase in both B _max and K _D. Changes in binding parameters for peripheral benzodiazepine receptors may be related to structural or permeability changes in mitochondrial outer membranes. In addition to the high-affinity (nanomolar range) binding site for peripheral benzodiazepine ligands, there is a low-affinity (micromolar range) saturable binding for PK 11195. At micromolar concentrations, peripheral benzodiazepines inhibit thiamine uptake by the cells. Altogether, our results suggest that impairment of oxidative metabolism, followed by mitochondrial swelling and disorganization of cristae, is the main cause of cell mortality in severely thiamine-deficient neuroblastoma cells.     

Human Y-79 Retinoblastoma Cells Exhibit Specific Corticotropin-Releasing Hormone Binding Sites

Abstract: In this study we have identified specific binding sites for corticotropin-releasing hormone (CRH) in human Y-79 retinoblastoma cell membranes by using ¹²⁵I-Tyrovine CRH (¹²⁵I-oCRH) as radioligand. Binding at 19°C was rapid with steady state being reached within 20 min, reversible and linear with membrane protein concentration. The ¹²⁵I-oCRH binding was enhanced by Mg²⁺ and inhibited by the GTP analogue guanosine 5'- O -(3'-thiotriphosphate). Y-79 cell membranes exhibited two populations of binding sites, a high-affinity site with an apparent dissociation constant ( K _D) of 1 n M and a low-affinity site with an apparent K _D of 500 n M . ¹²⁵I-oCRH binding was completely antagonized by human/rat CRH, [Met(O)²¹]oCRH, α-helical CRH_9–41, urotensin I, and sauvagine with a rank order of potency similar to that displayed by CRH receptors of other tissues. These data describe for the first time the presence of specific CRH-binding sites in retinal cells. The Y-79 cell line may therefore constitute a valuable model in which to study CRH action on retinal cells.     

Synthesis and SAR of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one as NMDA/glycine site antagonists

A series of 5-, 6-, 7- and 8-aza analogues of 3-aryl-4-hydroxyquinolin-2(1H)-one was synthesized and assayed as NMDA/glycine receptor antagonists. The in vitro potency of these antagonists was determined by displacement of the glycine site radioligand [(3)H]5,7-dicholorokynurenic acid ([(3)H]DCKA) in rat brain cortical membranes. Selected compounds were also tested for functional antagonism using electrophysiological assays in Xenopus oocytes expressing cloned NMDA receptor (NR) 1A/2C subunits. Among the 5-, 6-, 7-, and 8-aza-3-aryl-4-hydroxyquinoline-2(1H)-ones investigated, 5-aza-7-chloro-4-hydroxy-3-(3-phenoxyphenyl)quinolin-2-(1H)-one (13i) is the most potent antagonist, having an IC(50) value of 110 nM in [(3)H]DCKA binding and a K(b) of 11 nM in the electrophysiology assay. Compound 13i is also an active anticonvulsant when administered systemically in the mouse maximum electroshock-induced seizure test (ED(50)=2.3mg/kg, IP).