Intracellular calcium enhances the ethanol sensitivity of NMDA receptors through an interaction with the C0 domain of the NR1 subunit

Previous studies in this laboratory have shown that the ethanol inhibition of recombinant NMDA receptors expressed in Xenopus oocytes is subunit-dependent, with the NR1/2A receptor being more sensitive than NR1/2C receptors. The ethanol sensitivity of NR1/2A receptors is reduced by substitution of the wild-type NR1-1a (NR1(011)) subunit with the calcium-impermeable NR1 (N616R) subunit. In the present study, the ethanol inhibition of NMDA receptors was determined under different conditions to examine the role that calcium plays in determining the ethanol sensitivity of recombinant NMDA receptors. The ethanol sensitivity of NR1/2B or NR1/2C receptors was not affected by alterations in extracellular calcium levels or by coexpression with calcium-impermeable NR1 mutants. In contrast, the inhibition of NR1/2A receptors by 100 mM ethanol was reduced in divalent-free recording medium and was significantly increased when 10 mM calcium was used as the only charge carrier. The increase in the ethanol sensitivity of NR1/2A receptors under high-calcium conditions was prevented by preinjection of oocytes with the calcium chelator 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) but not by inhibitors of calmodulin or protein kinase C. Ethanol did not alter the channel blocking activity of divalent cations on NMDA-induced currents. The enhanced ethanol sensitivity of NR1/2A receptors in 10 mM calcium persisted when the NR1 subunit was replaced by the alternative splice variant NR1-4a (NR1(000)), which lacks the C1 and C2 cassettes. However, expression of a mutant NR1 subunit that lacked the C0, C1, and C2 domains abolished the calcium-dependent enhancement of ethanol's inhibition of NR1/2A receptors. Finally, the ethanol sensitivity of wild-type NR1/2A receptors measured in transfected HEK 293 cells by whole cell patch-clamp electrophysiology was significantly reduced by expression of the C-terminal truncated NR1 subunit. These results demonstrate that the ethanol sensitivity of certain NMDA receptors is modulated by an intracellular, calcium-dependent process that requires the C0 domain of the NR1 subunit.     

Protein kinase C modulation of recombinant NMDA receptor currents: roles for the C-terminal C1 exon and calcium ions

Protein kinase C (PKC) positively modulates NMDA receptor (NMDAR) currents. In contrast to previous reports, this study determines the importance of individual exons in the mechanism underlying the potentiation process by examining the complete set of eight naturally occurring splice variants expressed in Xenopus oocytes both as homomers and as heteromeric NR1/NR2A or NR1/NR2B complexes. After PKC stimulation, homomeric currents demonstrated a high level of potentiation ( approximately 500% of untreated baseline currents) that reduced to a lower level ( approximately 300% of baseline) in variants containing the first C-terminal exon (C1). An ANOVA showed that only C1 and no other exon or interaction of exons determined the degree of NMDAR current modulation by PKC. When recordings were performed in solutions in which barium replaces calcium, only the lower form of potentiation was observed, regardless of the splice variant exon composition. This suggested an important role for calcium in the PKC modulation of homomeric NMDA splice variant currents in which the C1 exon also participates. The effectiveness of the C1 exon to reduce the higher form of potentiation is modulated by heteromeric assemblies with NR2A heteromers yielding smaller levels of potentiation and a larger C1 exon effect compared with NR2B heteromers. The heteromers demonstrated the higher form of potentiation even in the absence of calcium. Furthermore, calcium had different effects in the potentiation of the heteromers depending on the NR2 subunit. This study refines the region of the NR1 subunit involved in a modulation crucial to the function of NMDA receptors and provides evidence that the NR2A and NR2B subunits realize this modulation differentially.     

Molecular dissection of native mammalian forebrain NMDA receptors containing the NR1 C2 exon: direct demonstration of NMDA receptors comprising NR1, NR2A, and NR2B subunits within the same complex

The subunit compositions of the NR1 C2 exon-containing N-methyl-D-aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.     

Lead inhibition of N-methyl-D-aspartate receptors containing NR2A, NR2C and NR2D subunits

The potency of Pb2+ inhibition of glutamate-activated currents mediated by N-methyl-D-aspartate (NMDA) receptors was dependent on the subunits composing the receptors when functionally expressed in Xenopus laevis oocytes. Pb2+ reduced the amplitudes of glutamate-activated currents and shifted the agonist EC50 values of NMDA receptors consisting of different subunit compositions. The IC50 values for Pb2+ ranged from 1.52 to 8.19 microM, with a rank order of potency of NR1b-2A > NR1b-2C > NR1b-2D > NR1b-2AC. For NR1b-2AC NMDA receptors, the IC50 value was dependent on the agonist concentration; at saturating agonist concentrations (300 microM), the IC50 value was 8.19 microM, whereas at 3 microM glutamate, the IC50 value was 3.39 microM. Pb2+ was a noncompetitive inhibitor of NR1b-2A, NR1b-2C and NR1b-2D NMDA receptors. At low concentrations (<1 microM) Pb2+ potentiated NR1b-2AC NMDA receptors. These data provide further evidence to support the hypothesis that the actions of Pb2+ on NMDA receptors are determined by the receptor subunit composition.     

Inhibition of calcium-dependent NMDA receptor current rundown by calbindin-D28k

NMDA receptors are regulated by several different calcium-dependent processes. To determine if the presence of the intracellular calcium-binding protein calbindin-D28k can influence the calcium regulation of NMDA receptor activity, human embryonic kidney 293 cells were co-transfected with cDNAs for NMDA receptor subunits and calbindin. Recordings were made using the nystatin perforated patch technique to preserve intracellular contents. When compared with control cells (transfected with cDNA encoding beta-galactosidase in place of calbindin), the presence of calbindin had no effect on either calcium-dependent inactivation or the calcium-sensitive, time-dependent increase in glycine-independent desensitization of NMDA receptor-mediated currents. However, the development of calcium-dependent rundown of peak glutamate-evoked current was slowed significantly in calbindin versus beta-galactosidase co-transfected cells. This result was true for cells transfected with either NR1/NR2A or NR1/NR2B subunits, although calbindin was relatively less effective at inhibiting rundown in NR1/NR2B-expressing cells. NMDA peak current rundown has been attributed to calcium-induced depolymerization of the actin cytoskeleton. Therefore, our results indicate that although calbindin may not influence calcium-dependent regulatory processes occurring very near the NMDA receptor channel, it appears to be more effective at buffering local elevations in intracellular calcium at the actin cytoskeleton.     

Identification of amino acid residues of the NR2A subunit that control glutamate potency in recombinant NR1/NR2A NMDA receptors

The NMDA type of ligand-gated glutamate receptor requires the presence of both glutamate and glycine for gating. These receptors are hetero-oligomers of NR1 and NR2 subunits. Previously it was thought that the binding sites for glycine and glutamate were formed by residues on the NR1 subunit. Indeed, it has been shown that the effects of glycine are controlled by residues on the NR1 subunit, and a "Venus flytrap" model for the glycine binding site has been suggested by analogy with bacterial periplasmic amino acid binding proteins. By analysis of 10 mutant NMDA receptors, we now show that residues on the NR2A subunit control glutamate potency in recombinant NR1/NR2A receptors, without affecting glycine potency. Furthermore, we provide evidence that, at least for some mutated residues, the reduced potency of glutamate cannot be explained by alteration of gating but has to be caused primarily by impairing the binding of the agonist to the resting state of the receptor. One NR2A mutant, NR2A(T671A), had an EC50 for glutamate 1000-fold greater than wild type and a 255-fold reduced affinity for APV, yet it had single-channel openings very similar to those of wild type. Therefore we propose that the glutamate binding site is located on NR2 subunits and (taking our data together with previous work) is not on the NR1 subunit. Our data further imply that each NMDA receptor subunit possesses a binding site for an agonist (glutamate or glycine).     

Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain

The NMDA (N-methyl D-aspartate) receptors in the brain play a critical role in synaptic plasticity, synaptogenesis and excitotoxicity. Molecular cloning has demonstrated that NMDA receptors consist of several homologous subunits (NMDAR1, 2A-2D). A variety of studies have suggested that protein phosphorylation of NMDA receptors may regulate their function and play a role in many forms of synaptic plasticity such as long-term potentiation. We have examined the phosphorylation of the NMDA receptor subunit NMDAR1 (NR1) by protein kinase C (PKC) in cells transiently expressing recombinant NR1 and in primary cultures of cortical neurons. PKC phosphorylation occurs on several distinct sites on the NR1 subunit. Most of these sites are contained within a single alternatively spliced exon in the C-terminal domain, which has previously been proposed to be on the extracellular side of the membrane. These results demonstrate that alternative splicing of the NR1 messenger RNA regulates its phosphorylation by PKC, and that mRNA splicing is a novel mechanism for regulating the sensitivity of glutamate receptors to protein phosphorylation. These results also provide evidence that the C-terminal domain of the NR1 protein is located intracellularly, suggesting that the proposed transmembrane topology model for glutamate receptors may be incorrect.     

NR2A subunit expression shortens NMDA receptor synaptic currents in developing neocortex

NMDA receptors play important roles in learning and memory and in sculpting neural connections during development. After the period of peak cortical plasticity, NMDA receptor-mediated EPSCs (NMDAR EPSCs) decrease in duration. A likely mechanism for this change in NMDA receptor properties is the molecular alteration of NMDA receptor structure by regulation of NMDA receptor subunit gene expression. The four modulatory NMDAR2A-D (NR2A-D) NMDA receptor subunits are known to alter NMDA receptor properties, and the expression of these subunits is regulated developmentally. It is unclear, however, how the four NR2 subunits are expressed in individual neurons and which NR2 subunits are important to the regulation of NMDA receptor properties during development in vivo. Analysis of NR2 subunit gene expression in single characterized neurons of postnatal neocortex revealed that cells expressing NR2A subunit mRNA had faster NMDAR EPSCs than cells not expressing this subunit, regardless of postnatal age. Expression of NR2A subunit mRNA in cortical neurons at even low levels seemed sufficient to alter the NMDA receptor time course. The proportion of cells expressing NR2A and displaying fast NMDAR EPSCs increased developmentally, thus providing a molecular basis for the developmental change in mean NMDAR EPSC duration.     

Calmodulin mediates calcium-dependent inactivation of N-methyl-D-aspartate receptors

Ca2+ influx through N-methyl-D-aspartate (NMDA) receptors activates signal transduction pathways critical for many forms of synaptic plasticity in the brain. NMDA receptor-mediated Ca2+ influx also downregulates the gating of NMDA channels through a process called Ca2+-dependent inactivation (CDI). Recent studies have demonstrated that the calcium binding protein calmodulin directly interacts with NMDA receptors, suggesting that calmodulin may play a role in CDI. We report here that the mutation of a specific calmodulin binding site in the CO region of the NR1 subunit of the NMDA receptor blocks CDI. Moreover, intracellular infusion of a calmodulin inhibitory peptide markedly reduces CDI of both recombinant and neuronal NMDA receptors. Furthermore, this inactivating effect of calmodulin can be prevented by coexpressing a region of the cytoskeletal protein alpha-actinin2 known to interact with the CO region of NR1. Taken together, these results demonstrate that the binding of Ca2+/calmodulin to NR1 mediates CDI of the NMDA receptor and suggest that inactivation occurs via Ca2+/calmodulin-dependent release of the receptor complex from the neuronal cytoskeleton.     

Characterization of protein kinase A and protein kinase C phosphorylation of the N-methyl-D-aspartate receptor NR1 subunit using phosphorylation site-specific antibodies

Modulation of N-methyl-D-aspartate receptors in the brain by protein phosphorylation may play a central role in the regulation of synaptic plasticity. To examine the phosphorylation of the NR1 subunit of N-methyl-D-aspartate receptors in situ, we have generated several polyclonal antibodies that recognize the NR1 subunit only when specific serine residues are phosphorylated. Using these antibodies, we demonstrate that protein kinase C (PKC) phosphorylates serine residues 890 and 896 and cAMP-dependent protein kinase (PKA) phosphorylates serine residue 897 of the NR1 subunit. Activation of PKC and PKA together lead to the simultaneous phosphorylation of neighboring serine residues 896 and 897. Phosphorylation of serine 890 by PKC results in the dispersion of surface-associated clusters of the NR1 subunit expressed in fibroblasts, while phosphorylation of serine 896 and 897 has no effect on the subcellular distribution of NR1. The PKC-induced redistribution of the NR1 subunit in cells occurs within minutes of serine 890 phosphorylation and reverses upon dephosphorylation. These results demonstrate that PKA and PKC phosphorylate distinct residues within a small region of the NR1 subunit and differentially affect the subcellular distribution of the NR1 subunit.     

High level expression of the NMDAR1 glutamate receptor subunit in electroporated COS cells

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 (KD = 29.6 +/- 6 nM; Bmax = 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-5H-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.     

Modulation of the N-methyl-D-aspartate receptor by haloperidol: NR2B-specific interactions

The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (epsilon2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high-affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (epislon1/epsilon2) receptors. Mutation of epsilon2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit 125I-MK-801 binding. Epsilon2E201 does not govern the interactions of ifenprodil, because all of the mutants at epsilon2E201 exhibited wild-type affinity for ifenprodil. Mutation of epsilon2R337 causes a 400-fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.     

Autophosphorylation-dependent targeting of calcium/ calmodulin-dependent protein kinase II by the NR2B subunit of the N-methyl- D-aspartate receptor

Activation and Thr286 autophosphorylation of calcium/calmodulindependent kinase II (CaMKII) following Ca2+ influx via N-methyl-D-aspartate (NMDA)-type glutamate receptors is essential for hippocampal long term potentiation (LTP), a widely investigated cellular model of learning and memory. Here, we show that NR2B, but not NR2A or NR1, subunits of NMDA receptors are responsible for autophosphorylation-dependent targeting of CaMKII. CaMKII and NMDA receptors colocalize in neuronal dendritic spines, and a CaMKII.NMDA receptor complex can be isolated from brain extracts. Autophosphorylation induces direct high-affinity binding of CaMKII to a 50 amino acid domain in the NR2B cytoplasmic tail; little or no binding is observed to NR2A and NR1 cytoplasmic tails. Specific colocalization of CaMKII with NR2B-containing NMDA receptors in transfected cells depends on receptor activation, Ca2+ influx, and Thr286 autophosphorylation. Translocation of CaMKII because of interaction with the NMDA receptor Ca2+ channel may potentiate kinase activity and provide exquisite spatial and temporal control of postsynaptic substrate phosphorylation.     

Block and modulation of N-methyl-D-aspartate receptors by polyamines and protons: role of amino acid residues in the transmembrane and pore-forming regions of NR1 and NR2 subunits

N-Methyl-D-aspartate (NMDA) receptors are modulated by extracellular spermine and protons and are blocked in a voltage-dependent manner by spermine and polyamine derivatives such as N1-dansyl-spermine (N1-DnsSpm). The effects of mutations in the first and third transmembrane domains (M1 and M3) and the pore-forming loop (M2) of NMDA receptor subunits were studied. Surprisingly, some mutations in M2 and M3 of the NR1 subunit, including mutations at W608 and N616 in M2, reduced spermine stimulation and proton inhibition. These mutations may have long-range allosteric effects or may change spermine- and pH-dependent gating processes rather than directly affecting the binding sites for these modulators because spermine stimulation and proton inhibition are not voltage dependent and are thought to involve binding sites outside the pore-forming regions of the receptor. A number of mutations in M1-M3, including mutations at tryptophan and tyrosine residues near the extracellular sides of M1 and M3, reduced block by spermine and N1-DnsSpm. The effects of these mutants on channel block were characterized in detail by using N1-DnsSpm, which produces block but not stimulation of NMDA receptors. Block by N1-DnsSpm was studied by using voltage ramps analyzed with the Woodhull model of channel block. Mutations at W563 (in M1) and E621 (immediately after M2) in the NR1A subunit and at Y646 (in M3) and N616 (in the M2 loop) in the NR2B subunit reduced the affinity for N1-DnsSpm without affecting the voltage dependence of block. These residues may form part of a binding site for N1-DnsSpm. Mutation of a tryptophan residue at position W607 in the M2 region of NR2B greatly reduced block by N1-DnsSpm, and N1-DnsSpm could easily permeate channels containing this mutation. The results suggest that at least parts of the M1 and M3 segments contribute to the pore or vestibule of the NMDA channel and that a tryptophan in M2 (W607 in NR2B) may contribute to the narrow constriction of the pore.     

Glutamate receptor ion channel properties predict vulnerability to cytotoxicity in a transfected nonneuronal cell line

Excessive activation of glutamate receptors is thought to play a critical role in neuronal excitotoxicity. To compare the cytotoxic potential of different glutamate receptor subtypes and correlate receptor biophysical properties with cytotoxicity, we have expressed recombinant receptors in human embryonic kidney 293 (HEK-293) cells. Survival of transfected cells was analyzed under conditions of defined agonist concentration and exposure time. For HEK-293 cells transfected with N-methyl-D-aspartate (NMDA) receptors, the EC50 for NMDA-induced cytotoxicity was 300 microM. Experiments using ion substitution, or cells expressing mutant NMDA receptors with low calcium permeability, suggested that both calcium and sodium influx through NMDA receptors contributed to cytotoxicity. In contrast, cytotoxicity was not observed in cells transfected with calcium permeable alpha-amino 3-hydroxy-5-methyl-4-isoxazole propionate- or kainate-type glutamate receptors even at saturating agonist concentrations, unless inhibitors of agonist-dependent desensitization were included. These results directly demonstrate that calcium permeability and desensitization kinetics play important roles in determining the excitotoxic potential of different glutamate receptor subtypes.     

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.     

Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro

The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.