Modulation of the N-Methyl-d-Aspartate Receptor by Haloperidol: NR2B-Specific Interactions

Abstract: The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (ε2) 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 (ε1/ε2) receptors. Mutation of ε2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit ¹²⁵I-MK-801 binding. ε2E201 does not govern the interactions of ifenprodil, because all of the mutants at ε2E201 exhibited wild-type affinity for ifenprodil. Mutation of ε2R337 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.     

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.     

Effects of Acute and Chronic Ethanol Exposure on Heteromeric N-Methyl-d-Aspartate Receptors Expressed in HEK 293 Cells

Abstract: Ion flux through native N-methyl-d -aspartate (NMDA) receptors is inhibited by behaviorally relevant concentrations of ethanol (10–100 mM) in a variety of neuronal preparations. However, in animal tissues, it is often difficult to determine accurately which NMDA receptor subunits are responsible for the observed effect. In this study, human embryonic kidney 293 (HEK 293) cells normally devoid of NMDA receptors were transiently transfected with cDNA expression plasmids coding for specific rat NMDA receptor subunits. Brief application of an NMDA/glycine solution to cells markedly increased intracellular calcium in cells transfected with NR1/NR2A, NR1/NR2B, or NR1/NR2A/NR2B as measured by fura-2 calcium imaging. This increase was both NMDA- and glycine-dependent and was inhibited by competitive and noncompetitive NMDA antagonists, including 2-amino-5-phosphopentanoic acid and MK-801. The NR2B-selective antagonist ifenprodil inhibited responses in cells transfected with NR1/NR2B or NR1/NR2A/NR2B, but not NR1/NR2A subunits. Increasing the transfection ratio of NR2B versus NR2A subunit in NR1/NR2A/NR2B-transfected cells greatly increased their ifenprodil sensitivity. Acute exposure to ethanol (25–100 mM) inhibited the NMDA-mediated increase in intracellular calcium in a dose-dependent manner without affecting basal calcium concentrations. There were no statistically significant differences in ethanol's potency or maximal inhibition between any of the subunit combinations tested. HEK 293 cells transfected with NR1/NR2A/NR2B subunits showed an enhanced sensitivity to ifenprodil following a 24-h exposure to concentrations of ethanol of 50 mM and greater. The enhanced ifenprodil sensitivity following ethanol exposure was not associated with changes in NR1, NR2A, or NR2B immunoreactivity. In contrast to results obtained in transfected HEK 293 cells, no effect of chronic ethanol was observed in oocytes expressing NR1/NR2A/NR2B subunits. These results demonstrate that recombinant NMDA receptors expressed in HEK 293 cells form functional receptors that, like native receptors, are sensitive to modulation by both acute and chronic ethanol treatment.     

Inducible expression and pharmacological characterization of recombinant rat NR1a/NR2A NMDA receptors

In this study, we have established a non-neuronal cell line stably and inducibly expressing recombinant NMDA receptors (NRs) composed of rat NR1a/NR2A subunits. EcR-293 cells were transfected with rat NR1a and NR2A cDNAs using the inducible mammalian expression vector pIND. Cell colonies resistant for the selecting agents were picked and tested for NR2A mRNA as well as protein expression using quantitative RT-PCR and flow cytometry based immunocytochemistry. Clonal cells expressing functional NMDA receptors were identified by measuring NMDA-evoked ion currents, and NMDA-induced increase in cytosolic free calcium concentration in whole-cell patch-clamp and fluorimetric calcium measurements, respectively. One clone named D5/H3, which exhibited the highest response to NMDA, was chosen to examine inducibility of the expression and for pharmacological profiling of recombinant NR1a/NR2A NMDA receptors. To check inducibility, NR2A subunit expression in D5/H3 cells treated with the inducing agent muristerone A (MuA) was compared with that in non-induced cells. Both NR2A mRNA and protein expression was several folds higher in cells treated with the inducing agent. As part of the pharmacological characterization, we examined the activation of the expressed NR1a/NR2A receptors as a function of increasing concentration of NMDA. NMDA-evoked concentration-dependent increases in cytosolic [Ca2+] with an EC50 value of 41 +/- 1 microM. In addition, whereas the NMDA response was concentration-dependently inhibited by the channel blocker MK-801 (IC50 = 58 +/- 6 nM), NR2B subunit selective NMDA receptor antagonists were ineffective. Thus, this cell line, which stably and inducibly expresses recombinant NR1a/NR2A NMDA receptors, can be a useful tool for testing NMDA receptor antagonists and studying their subunit selectivity.     

Biochemical Evidence for the Existence of a Pool of Unassembled C2 Exon-Containing NR1 Subunits of the Mammalian Forebrain NMDA Receptor

Abstract: Optimum conditions were determined for the solubilisation of native NMDA receptors of adult mammalian brain with the retention of [³H]MK-801 radioligand binding activity. The most efficient conditions were 1% Triton X-100/1 M NaCl. The efficiency of solubilisation was as follows: cloned NMDA receptors expressed in mammalian cells > forebrain receptors > cerebellar receptors. Triton X-100/1 M NaCl-solubilised forebrain NMDA receptors had a molecular size of 710,000 daltons, but significant NR1 immunoreactivity (41%) migrated as a monomer of 125,000 daltons. Immunoaffinity purification of NMDA receptors from forebrain by anti-NR1 911–920 antibody affinity chromatography from 1% Triton X-100/1 M NaCl solubilised extracts yielded purification of the NR1 M_r 120,000 immunoreactive species, but no detectable NR2A or NR2B immunoreactivity. Immunoprecipitation of NMDA receptors from Triton X-100/1 M NaCl extracts with anti-NR1 911–920 antibodies also resulted in precipitation of NR1 subunits, but with no detectable NR2A or NR2B subunits. In contrast, by immunoprecipitation with anti-NR1 17–35 antibodies, which recognise all forms of NR1, NR1, NR2A, and NR2B immunoreactivities were detected in the immune pellets. Similarly, a coassociation of NR1, NR2A, and NR2B subunits was demonstrated following extraction of forebrain membranes with 1% sodium deoxycholate (pH 9) and purification by anti-NR1 911–920 antibody affinity chromatography. These results are consistent with the identification of a pool of unassembled C2 exon-containing NR1 subunits, i.e., NR1-1a, NR1-1b, NR1-2a, and NR1-2b, selectively solubilised by 1% Triton X-100/1 M NaCl.     

Cloning, Functional Coexpression, and Pharmacological Characterisation of Human cDNAs Encoding NMDA Receptor NR1 and NR2A Subunits

Abstract: 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 μ M for glutamate and glycine, respectively, and an IC₅₀ of 46.8 μ M 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.     

[3H]Homoquinolinate Binds to a Subpopulation of NMDA Receptors and to a Novel Binding Site

Abstract: NMDA receptors mediate several important functions in the CNS; however, little is known about the pharmacology, biochemistry, and function of distinct NMDA receptor subtypes in brain tissue. To facilitate the study of native NMDA receptor subpopulations, we have determined the radioligand binding properties of [³H]homoquinolinate, a potential subtype-selective ligand. Using quantitative receptor autoradiography, NMDA-specific [³H]homoquinolinate binding selectively labeled brain regions expressing NR2B mRNA (layers I–III of cerebral cortex, striatum, hippocampus, and septum). NMDA-specific [³H]homoquinolinate binding was low in brain regions that express NR2C and NR2D mRNA (cerebellar granular cell layer, NR2C; glomerular layer of olfactory bulb, NR2C/NR2D; and midline thalamic nuclei, NR2D). In forebrain, the pattern of NMDA-specific [³H]homoquinolinate binding paralleled NR2B and not NR2A distribution. In addition to NMDA-displaceable binding, there was a subpopulation of [³H]homoquinolinate binding sites in the forebrain, cerebellum, and choroid plexus that was not displaced by NMDA or l -glutamate. In contrast, we found that the derivative of homoquinolinate, 2-carboxy-3-carboxymethylquinoline, markedly inhibited the NMDA-insensitive binding of [³H]homoquinolinate without inhibiting the NMDA-sensitive population. [³H]Homoquinolinate may be useful for selectively characterizing NR2B-containing NMDA receptors in a preparation containing multiple receptor subtypes and for characterizing a novel binding site of unknown function.     

Neurosteroids allosterically modulate the ion pore of the NMDA receptor consisting of NR1/NR2B but not NR1/NR2A

Neurosteroids are endogenously derived compounds, mediating rapid effects in the central nervous system. They participate in vital processes, including memory and learning, neuroplasticity, and neuroprotection in Alzheimer’s disease. However, the mechanisms behind those effects remain to be elucidated. The neurosteroids pregnenolone sulphate (PS) and pregnanolone sulphate (3α5βS) have recently been shown to allosterically alter the NMDA receptor in nanomolar concentrations. Those studies featured ifenprodil, which is a dirty drug, with affinity to many targets. In this study we compare the NMDA receptors in the hippocampus to recombinant NMDA receptors, using [³H]-MK-801 as radioligand. The results show that neurosteroids modulate the ifenprodil binding kinetics in a narrow concentration interval, addressing it to the NR2B subunit, since no effects were recorded at recombinant NR1/NR2A receptors. The effects were also seen as changes in the manner ifenprodil displaced or induced the dissociation of [³H]-MK-801. It indicates that the neurosteroidal effects indeed alter the ion pore of the NMDA receptor, why it is reasonable to believe that these findings have physiological relevance.     

Modulation of 45Ca2+ Influx into Cells Stably Expressing Recombinant Human NMDA Receptors by Ligands Acting at Distinct Recognition Sites

Abstract: A ⁴⁵Ca²⁺ influx assay has been used to investigate the pharmacology of stably expressed recombinant human NR1a/NR2A and NR1a/NR2B N -methyl- d -aspartate (NMDA) receptors. Inhibition of glutamate-stimulated ⁴⁵Ca²⁺ influx by six glycine-site antagonists and inhibition of glycine-stimulated ⁴⁵Ca²⁺ influx by five glutamate-site antagonists revealed no significant differences between affinity values obtained for NR1a/NR2A and NR1a/NR2B receptors. The polyamine site agonist spermine showed differential modulation of glutamate- and glycine-stimulated ⁴⁵Ca²⁺ influx for recombinant NMDA receptors, inhibiting and stimulating ⁴⁵Ca²⁺ influx into cells expressing NR1a/NR2A receptors (IC₅₀ = 408 µ M ) and NR1a/NR2B receptors (EC₅₀ = 37.3 µ M ), respectively. The antagonist ifenprodil was selective for NR1a/NR2B receptors (IC₅₀ = 0.099 µ M ) compared with NR1a/NR2A receptors (IC₅₀ = 164 µ M ). The effects of putative polyamine site antagonists, redox agents, ethanol, and Mg²⁺ and Zn²⁺ ions were also compared between NR1a/NR2A and NR1a/NR2B receptors. This study demonstrates the use of ⁴⁵Ca²⁺ influx as a method for investigating the pharmacology of the numerous modulatory sites that regulate the function of recombinant human NMDA receptors stably expressed in L(tk-) cells.     

Presynaptic NMDA Receptors Display Physiological Characteristics of Homomeric Complexes of NR1 Subunits that Contain the Exon 5 Insert in the N-Terminal Domain

Abstract: The subunit composition of the N -methyl- d -aspartate (NMDA) glutamate receptor affects both its channel activity and its sensitivity to modulation by a wide variety of substances. Expression studies in oocytes and physiological studies in neurons indicate that endogenous postsynaptic NMDA receptors are heterooligomeric complexes of NR1 and NR2 subunits. To deduce the subunit composition of the presynaptic NMDA receptor on noradrenergic nerve terminals, we examined the modulation of NMDA-evoked norepinephrine (NE) release from hippocampal synaptosomes. At high glycine concentrations, the NMDA-evoked release was not potentiated by reducing reagents, low micromolar Zn²⁺ or Ni²⁺, polyamines, or 100 µ M histamine. It was also not inhibited by oxidizing agents or physiological concentrations of protons but was inhibited by high micromolar Co²⁺, Zn²⁺, and Ni²⁺, but not Fe³⁺, by high micromolar ifenprodil, and by 1 m M histamine. At low glycine concentrations, it was potentiated by spermine. These characteristics are similar to those displayed by homooligomeric complexes of NR1 subunits that contain in the N-terminal domain the 21-amino-acid insert encoded by exon 5. These data provide physiological evidence that some endogenous NMDA receptor complexes may contain only the NR1 (+ exon 5) subunits.     

Regionally Distinct N-Methyl-D-Aspartate Receptors Distinguished by Quantitative Autoradiography of [3H]MK-801 Binding in Rat Brain

Abstract: Quantitative autoradiography of [³H]MK-801 binding was used to characterize regional differences in N -methyl- d -aspartate (NMDA) receptor pharmacology in rat CNS. Regionally distinct populations of NMDA receptors were distinguished on the basis of regulation of [³H]MK-801 binding by the NMDA antagonist 3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP). CPP inhibited [³H]MK-801 binding in outer cortex (OC) and medial cortex (MC) with apparent K _i values of 0.32-0.48 μ M , whereas in the medial striatum (MS), lateral striatum (LS), CA1, and dentate gyrus (DG) of hippocampus, apparent K _i values were 1.1-1.6 μ M . In medial thalamus (MT) and lateral thalamus (LT) the apparent K _i values were 0.78 μ M . In the presence of added glutamate (3 μ M ), the relative differences in apparent K _i values between regions maintained a similar relationship with the exception of the OC. Inhibition of [³H]MK-801 binding by the glycine site antagonist 7-chlorokynurenic acid (7-ClKyn) distinguished at least two populations of NMDA receptors that differed from populations defined by CPP displacement. 7-ClKyn inhibited [³H]MK-801 binding in OC, MC, MS, and LS with apparent K _i values of 6.3-8.6 μ M , whereas in CA1, DG, LT, and MT, K _i values were 11.4-13.6 μ M . In the presence of added glycine (1 μ M ), the relative differences in apparent K _i values were maintained. Under conditions of differential receptor activation, regional differences in NMDA receptor pharmacology can be detected using [³H]MK-801 binding.     

Characterisation of N-methyl-D-aspartate receptor-specific [(3)H]Ifenprodil binding to recombinant human NR1a/NR2B receptors compared with native receptors in rodent brain membranes

We have performed [(3)H]ifenprodil binding experiments under NMDA receptor-specific assay conditions to provide the first detailed characterisation of the pharmacology of the ifenprodil site on NMDA NR1/NR2B receptors, using recombinant human NR1a/NR2B receptors stably expressed in L(tk-) cells, in comparison with rat cortex/hippocampus membranes. [(3)H]Ifenprodil bound to a single, saturable site on both human recombinant NR1a/NR2B receptors and native rat receptors with B:(max) values of 1.83 and 2.45 pmol/mg of protein, respectively, and K:(D) values of 33.5 and 24.8 nM:, respectively. The affinity of various ifenprodil site ligands-eliprodil, (R:(*), R:(*))-4-hydroxy-alpha-(4-hydroxyphenyl)-beta-methyl-4-pehnyl-1-pi per idineethanol [(+/-)-CP-101,606], cis-3-[4-(4-fluorophenyl)-4-hydroxy-1-piperidinyl]-3, 4-dihydro-2H:-1-benzopyran-4,7-diol [(+/-)-CP-283,097], and (R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperid inepropanol [(+/-)-Ro 25-6981] was very similar for inhibition of [(3)H]ifenprodil binding to recombinant human NR1a/NR2B and native rat receptors, whereas allosteric inhibition of [(3)H]ifenprodil binding by polyamine site ligands (spermine, spermidine, and arcaine) showed approximately twofold lower affinity for recombinant receptors compared with native receptors. Glutamate site ligands were less effective at modulating [(3)H]ifenprodil binding to recombinant NR1a/NR2B receptors compared with native rat receptors. The NMDA receptor-specific [(3)H]ifenprodil binding conditions described were also applied to ex vivo experiments to determine the receptor occupancy of ifenprodil site ligands [ifenprodil, (+/-)-CP-101,606, (+/-)-CP-283,097, and (+/-)-Ro 25-6981] given systemically.     

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.     

Opposing Contributions of NR1 and NR2 to Protein Kinase C Modulation of NMDA Receptors

Abstract: N -Methyl- d -aspartate (NMDA) receptors mediate increases in intracellular calcium that can be modulated by protein kinase C (PKC). As PKC modulation of NMDA receptors in neurons is complex, we studied the effects of PKC activation on recombinant NMDA receptor-mediated calcium rises in a nonneuronal mammalian cell line, human embryonic kidney 293 (HEK-293). Phorbol 12-myristate 13-acetate (PMA) pretreatment of HEK-293 cells enhanced or suppressed NMDA receptor-mediated calcium rises based on the NMDA receptor subunit composition. NR2A or NR2B, in combination with NR1₀₁₁, conveyed enhancement whereas NR2C and NR2D conveyed suppression. The PKC inhibitor bisindolylmaleimide blocked each of these effects. The region on NR2A that conveyed enhancement localized to a discrete segment of the C terminus distal to the portion of NR2C that is homologous to NR2A. Calcium-45 accumulation, but not intracellular calcium store depletion, matched PMA effects on NMDA receptor-mediated calcium changes, suggesting that these effects were not due to effects on intracellular calcium stores. The suppression of intracellular calcium transients seen with NR2C was eliminated when combined with NR1 splice variants lacking C-terminal cassette 1. Thus, the intracellular calcium effects of PMA were distinguishable based on both the NR1 splice variant and the NR2 subunit type that were expressed. Such differential effects resemble the diversity of PKC effects on NMDA receptors in neurons.     

Inducible expression and pharmacology of recombinant NMDA receptors,composed of rat NR1a/NR2B subunits

An ecdysone-inducible mammalian expression system was used to study expression of recombinant N-methyl-D-aspartate (NMDA) receptors. Human embryonic kidney (HEK) 293 cells expressing the regulatory vector pVgRXR (EcR 293 cells) were transfected with rat NR1a and NR2B cDNAs using the inducible vector pIND (Invitrogen). Inducible expression of the NR2B subunit in cell clone designated EcR/rNR1a2B was investigated using quantitative RT-PCR and flow cytometry based immunocytochemical methods. The mRNA level of the NR2B subunits in EcR/rNRa2B cells was dependent on the concentration of the ecdysone analogue inducing agent, muristerone A (MuA). Similarly, NR2B subunit protein expression was higher in cells pre-treated with the inducing agent. Functionally active NMDA receptors were also detected in EcR/rNR1a2B cells after MuA induction. In presence of the inducing factor, NMDA-evoked ion currents as well as increase in cytoplasmic calcium-concentrations were measured using whole-cell patch clamp and fluorometric calcium measuring techniques. The pharmacological profile of the expressed NMDA receptors was characterised by comparing the inhibitory activity of several NR2B subunit selective NMDA antagonists in EcR/rNR1a2B cells with that observed in primary cultures of rat cortical neurones. Whereas the efficacies of the NR2B subunit selective NMDA antagonists were similar in EcR/rNR1a2B cells and in neurones, their maximal inhibitory effects were significantly higher in cells expressing NR1a/NR2B recombinant receptors. This study demonstrates that recombinant NMDA receptors can be expressed in an inducible way in non-neuronal cell lines using the ecdysone-inducible mammalian expression system. Such cell lines can be suitable tools in high throughput functional screening for potential subtype selective modulators of the NMDA receptor.     

Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Up-Regulation of NR2B Subunit of NMDA Receptors in Cerebellar Granule Neurons by Ca2+/Calmodulin Kinase Inhibitor KN93

Abstract: Recordings of NMDA-activated currents from cerebellar granule neurons in culture revealed a developmental increase in current density accompanied by a slight decrease of the half-maximal effective concentration. At the same time, a decrease of NMDA receptors comprising NR2B subunits was demonstrated by the reduction in the antagonism of NMDA currents by ifenprodil. Ifenprodil antagonism increased after treatment for 24 h with KN93- and KN62-selective inhibitors of the Ca²⁺/calmodulin-dependent protein kinases (CaM kinases), indicating a selective increase of receptor containing NR2B subunit. This increase was observed at all ages tested: 4 days in vitro (DIV4), DIV6, and DIV13. Western blot analysis with specific NMDA receptor antibodies performed at DIV6 confirmed the electrophysiological data. At this age, the negative control KN92 was ineffective. The increasing ifenprodil antagonism after KN93 treatment was proportionally greater in cells at DIV13 than at DIV4. Treatment with NMDA (100 µ M ) of cerebellar cultures for 24 h produced a decrease in the NMDA-induced current density by almost 50% at all ages tested. Ifenprodil antagonism, however, was unchanged. We propose that the expression of NR2B subunits in cerebellar granule cells is selectively stimulated by the inhibition of CaM kinases.     

Toxicity of beta-amyloid in HEK293 cells expressing NR1/NR2A or NR1/NR2B N-methyl-D-aspartate receptor subunits

Neurotoxicity induced by beta-amyloid peptide (Abeta) involves glutamate toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) receptors and elevation of intracellular calcium. However, the heterogeneity of the NMDA receptors, frequently composed of NR1 and NR2A-D subunits, has been less studied. Thus, we determined the contribution of NMDA receptor subtypes on Abeta(1-40) toxicity in HEK293 cells transiently expressing NR1/NR2A or NR1/NR2B subunits. Analysis of lactate dehydrogenase (LDH) release and trypan blue exclusion revealed an increase in Abeta(1-40) toxicity upon NR1/NR2A expression, compared to NR1/NR2B, indicating loss of plasma membrane integrity. Furthermore, Abeta(1-40) decreased intracellular ATP in cells expressing NR1/NR2A. MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate), a noncompetitive NMDA receptor antagonist, partially prevented the decrease in cell viability and the energy impairment. These differences were not accounted for by the activation of caspases 2, 3, 8 and 9 or calpains or by DNA fragmentation, excluding the hypothesis of apoptosis. Functional NR1/NR2A and NR1/NR2B receptor subtypes were further evidenced by single-cell calcium imaging. Stimulation of NR1/NR2A receptors with NMDA/glycine revealed an increase in intracellular calcium in cells pre-exposed to Abeta(1-40). Opposite effects were observed upon activation of NR1/NR2B receptors. These results suggest that NR1/NR2A-composed NMDA receptors mediate necrotic cell death in HEK293 cells exposed to Abeta(1-40) through changes in calcium homeostasis.     

Differential Profiles of Binding of a Radiolabeled Agonist and Antagonist at a Glycine Recognition Domain on the N- Methyl-D-Aspartate Receptor lonophore Complex in Rat Brain

Abstract: Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [³H] 5, 7-dichlorokynurenic acid ([³H]- DCKA) but not of the agonist ligand [³H] glycine ([³H] Gly) to a Gly recognition domain on the N -methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [³H] DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (±)-α-(4-chlorophenyl)-4- [(4-fluorophenyl)methyl]-1-piperidine ethanol, with [³H] Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [³H] DCKA binding virtually without affecting those of four different agonists. Phospholipases A₂ and C and p -chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [³H] DCKA binding with [³H] Gly binding being unaltered. Moreover, the densities of [³H] DCKA binding were not significantly different from those of [³H]- Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [³H] Gly binding than of [³H] DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.