Decreased expression and functionality of NMDA receptor complexes persist in the CA1, but not in the dentate gyrus after transient cerebral ischemia

The authors investigated the gene expression of the NR2A and NR2B subunits of N-methyl-D-aspartate (NMDA) receptor and the functional electrophysiologic activity of NMDA receptor complexes in the vulnerable CA1 and less vulnerable dentate gyrus subfields of the rat hippocampus at different times after transient cerebral ischemia. Decreased expression for both subtypes was observed in both the CA1 subfield and dentate granule cell layer at early times after challenge; however, the decreased expression in the dentate granule cell layer was reversible because mRNA levels for both the NR2A and NR2B subtypes recovered to, or surpassed, sham-operated mRNA levels by 3 days postchallenge. No recovery of expression for either subtype was observed in the CA1 subfield. The functional activity of NMDA receptor complexes, as assessed by slow field excitatory postsynaptic potentiations (slow f-EPSP) in CA1 pyramidal neurons, was maintained at 6 hours postchallenge; however, this activity was diminished greatly by 24 hours postchallenge, and absent at 7 days postchallenge. A similar pattern was observed for the non-NMDA receptor-mediated fast f-EPSP. In dentate granule neurons, however, no significant change in NMDA receptor-mediated slow f-EPSP from sham control was observed at any time after insult. The non-NMDA receptor-generated fast f-EPSPs also were maintained at all times postinsult in the dentate gyrus. These results illustrate that the activity of NMDA receptors remains functional in dentate granule neurons, but not in the pyramidal neurons of the CA1 subfield, at early and intermediate times after transient cerebral ischemia, and suggest that there is a differential effect of ischemia on the glutamatergic transmission systems in these two hippocampal subfields.     

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).     

Endogenous glutamate levels regulate nerve growth factor mRNA expression in the rat dentate gyrus

The levels of nerve growth factor (NGF) mRNA can be regulated in vitro and in vivo in the hippocampal formation by events associated with pharmacological activation of glutamate receptors. In the present study, the level of NGF mRNA in the hippocampal formation was examined following an intrahippocampal injection of 1 nmole fluorocitrate, which temporarily inhibits the astrocyte metabolic activity in vivo. Consistent with previous findings, fluorocitrate treatment significantly increased glutamate levels and decreased glutamine levels in the dentate gyrus as determined by in vivo microdialysis. The increased ratio of glutamate to glutamine was followed by a significant increase in NGF mRNA expression selectively in dentate gyrus granule cells. The effects of increasing glutamate levels were blocked by pretreatment with 50 nmole 2-amino-5-phosphonovalerate (AP5), a competitive antagonist that acts at the N-methyl-D-aspartate (NMDA) glutamate receptor subtype. These findings suggest that NGF mRNA expression is regulated, in part, by changes in endogenous glutamate levels, partially through enhanced excitatory neurotransmission through NMDA receptors.     

Increase in syntaxin 1B and glutamate release in mossy fibre terminals following induction of LTP in the dentate gyrus: a candidate molecular mechanism underlying transsynaptic plasticity

A growing body of evidence suggests that modulation of certain proteins of the exocytotic machinery is, in part, involved in the biochemical changes that underlie long-term synaptic plasticity. We have previously shown that the induction of long-term potentiation (LTP) at perforant path to dentate granule cell synapses in the rat hippocampus induces changes in the mRNA levels of syntaxin 1B and synapsin I, known to be involved in neurotransmitter release. Immunohistochemical staining suggested that concomitant changes in these proteins occurred at mossy fibre synapses, downstream of those synapses at which LTP was induced, leading us to postulate that such a mechanism might underlie a form of transsynaptic plasticity. Here we have used a specific mossy-fibre synaptosome preparation to quantify levels of proteins and measure, using a chemiluminescent glutamate assay, depolarization-induced glutamate release from these synaptosomes after induction of LTP in the dentate gyrus in vivo. We show that 5 h after the induction of LTP, there is an increase in the protein levels of syntaxin 1B and, although to a lesser extent, the synapsins I and II, associated with an increase in depolarization-induced release of glutamate within these terminals. Increases in both the protein levels and glutamate release were not observed when dentate gyrus LTP was blocked by an NMDA receptor antagonist. From these results we propose a molecular mechanism for the propagation of synaptic plasticity through hippocampal circuits.     

NMDA-NR1 receptor subunit mRNA expression in rat brain after 6-OH-dopamine induced lesions: a non-isotopic in situ hybridization study

Antisense digoxigenin-labeled deoxyoligonucleotides probes and non-isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA-NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6-OH-dopamine (6-OH-DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1-CA2-CA3 of the hippocampus, in layers II-III and V-VI of the cerebral cortex, and in the cerebellum of sham-treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6-OH-DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6-OH-DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6-OH-DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6-OH-DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA-containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamateric neurons and the genic expression of NMDA receptors.     

Long-term potentiation in vivo increases rat hippocampal tenascin-C expression

We investigated the expression of the extracellular matrix glycoprotein tenascin-C after induction of long-term potentiation (LTP) by high-frequency tetanization (HFT) in the rat dentate gyrus in vivo. Expression of tenascin-C was evaluated at the mRNA and protein levels by in situ hybridization and immunocytochemistry, respectively. Whereas no tenascin-C mRNA was detectable in control animals, an increase in tenascin-C mRNA levels was observed in the granule cell layer of the dentate gyrus 4 h after HFT. At 24 h after HFT, tenascin-C mRNA had returned to control levels. Expression of tenascin-C protein 4 h after HFT followed that of controls in that tenascin was detectable in the strata oriens and radiatum of CA1, in the molecular layer, and within a narrow area at the inner surface of the granule cell layer in the dentate gyrus. However, 24 h after HFT, additional patches of tenascin-C immunoreactivity were observed in the molecular layer of the dentate gyrus. No increase in tenascin mRNA or protein levels was detected in control animals that received no stimulation, low-frequency stimulation, or HFT in the presence of the N-methyl-D-aspartate receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid or the metabotropic glutamate receptor antagonist (R,S)-alpha-methyl-4-carboxyphenylglycine. These observations implicate a role for tenascin-C in N-methyl-D-aspartate and metabotropic glutamate receptor-dependent changes accompanying induction and/or maintenance of LTP.     

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.     

Adjacent guanines as preferred sites for strand breaks in plasmid DNA irradiated with 193 nm and 248 nm UV laser light

Kainate-preferring glutamate receptors may contribute to the glutamatergic responses to seizures. The cloning of their encoding genes overcomes limitations of the receptor ligands available for their investigation. We have examined the expression of the high affinity kainate receptor subunits KA1 and KA2 mRNAs in the rat hippocampus, using electroconvulsive shock (ECS) as a seizure paradigm not confounded by neurotoxicity. A single shock reduced the levels of KA1 mRNA in the CA3c region, while increasing the expression of KA2 mRNA in the dentate gyrus. Following repeated ECS (5 shocks over 10 days), KA1 mRNA was reduced in CA3c and in CA3a-b but was unchanged in dentate gyrus. KA2 mRNA, on the other hand, significantly increased in dentate gyrus, and to a lesser extent in CA3c and CA1. All changes in KA1 and KA2 mRNAs had returned to baseline 3 weeks after the last shock. We also measured the expression of cyclophilin mRNA, and found it to be reduced in all hippocampal subfields, and in the parietal cortex, after a single ECS. It returned to control levels after repeated ECS but was again reduced following 3 weeks recovery from repeated ECS. These results indicate that the expression of KA1 and KA2 not only change in opposite directions in the rat hippocampus after ECS, but that the alterations are anatomically and temporally regulated. In the respect that cyclophilin is regarded as a housekeeping gene, the reduction in its mRNA suggests that ECS may have more persistent and widespread effects on brain gene expression than previously suspected.     

Dopamine receptor sensitive effect of dizocilpine on feeding behaviour

The lactating rat has been shown to lack a behavioral response and immediate early gene expression (cFos) in the hippocampus (Hipc) following intravenous or intracerebroventricular administration of an N-methyl-d-aspartate (NMDA) receptor agonist. The purpose of this study is to determine whether neurons in the Hipc have an intact postsynaptic NMDA receptor system. The presence of NMDA receptor protein was determined by Western blot analysis for the NR1, NR2A, and NR2B subunits. The presence of functional NMDA receptors in the Hipc was determined by behavioral responses and the expression of cFos immunoreactivity (-ir) in response to microinjection of an NMDA receptor agonist into the hilus of the dentate gyrus. No difference in NR1 and NR2A subunit protein in the Hipc was detected between the lactating and nonlactating rats. However, there was a 26% decrease in NR2B subunit protein in this region in the lactating rat. Lactating rats receiving NMA injections displayed hyperactive behavior, similar to that observed in the nonlactating animals receiving the same treatment. The lactating rat and the nonlactating rat also displayed equivalent bilateral cFos-ir in the dentate gyrus (DG), CA1 and CA3 regions of the Hipc in response to unilateral NMA injections into the Hipc. These data indicate that the lactating rat has an intact postsynaptic NMDA receptor system. Thus, Hipc refractoriness to peripheral and third ventricular injections of an NMDA receptor agonist may reflect inhibition of presynaptic input and glutamate release.     

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.     

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.     

Opposing contributions of NR1 and NR2 to protein kinase C modulation of NMDA receptors

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 NR1(011), 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.     

Immunoblot analyses on the differential distribution of NR2A and NR2B subunits in the adult rat brain

Quantitative immunoblot analyses were carried out to study the distribution of N-methyl-D-aspartate (NMDA) receptor subunit 2A and 2B (NR2A and NR2B, respectively) at the protein level in the adult rat brain. Highest levels of NR2A were detected in cerebral cortex and hippocampus, followed at more or less similar levels (about 36-72% of cerebral cortex) by striatum, thalamus, olfactory bulb, superior and inferior colliculi, and cerebellum. The lowest levels were detected in midbrain and lower brain stem (30-31% of cerebral cortex). The NR2B was more dramatic in differential distribution than the NR2A. Highest levels of NR2B were found in telencephalic (olfactory bulb, cerebral cortex, hippocampus, and striatum) and thalamic regions, and expression in superior and inferior colliculi, midbrain, lower brain stem, and cerebellum were significantly lower (4-25% of cerebral cortex). Interestingly, NR2B proteins were barely detectable in the cerebellum. When the postsynaptic density (PSD) fractions were compared, the amount of NR2B in the cerebellar PSD fraction was only 1.8% of that present in the cerebral PSD fraction where the subunit is highly enriched. Immunoblot analyses with a phosphotyrosine-specific antibody showed that the molecular sizes of major phosphotyrosine-containing proteins in forebrain and hindbrain are 180 and 45 kDa, respectively. The regional distribution of the 180 kDa major phosphotyrosine protein was very similar to that of NR2B, and the protein could be immunoprecipitated by NR2B antibody. Our data shows that NR2A and NR2B subunits are differentially distributed in the brain in an overlapping manner, and that the major phosphotyrosine-containing protein of 180 kDa in forebrain is the NR2B.     

The NMDA receptor subunit NR2B of neonatal rat brain: complex formation and enrichment in axonal growth cones

The N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors comprises a family of highly homologous subunits which assemble into oligomeric protein complexes. Alterations in subunit composition are developmentally regulated, leading to functionally distinct receptor populations. Here, the contribution of the subunit NR2B to NMDA receptor complex formation was analysed in neonatal rat brain, employing polyclonal antibodies raised against NR2B-specific synthetic peptides. By hydrodynamic size fractionation of the solubilized receptor protein and chemical cross-linking, NR2B antigen was found to be associated with several protein species of up to 690 kDa molecular weight. These observations show NR2B to be part of a multimeric receptor complex. Fractionation of cortex homogenates from E18 rat embryos on sucrose density gradients revealed NR2B polypeptide to be highly enriched in axonal growth cones. A similar distribution was found by fluorescence microscopy of immature hippocampal neurons, showing a preferential accumulation of NR2B antigen in axonal growth cones and varicosities. In mature cells, NR2B antigen displayed a punctated distribution pattern with redistribution to somato-dendritic spheres. The association of NR2B with axonal growth cones and processes of immature neurons suggests a role of NMDA receptors in the regulation of neurite outgrowth and migration.     

Long-lasting enhanced expression in the rat hippocampus of NMDAR1 splice variants in a kainate model of epilepsy

Chronic epilepsy is associated with increased excitability which may result from abnormal glutamatergic synaptic transmission involving altered properties of N-methyl-D-aspartate (NMDA) receptors. To date two gene families encoding NMDA receptor subunits have been cloned, NR1 and NR2. Eight NR1 mRNAs are generated by alternative splicing of exons 5, 21 and 22; the NR1-1 to NR1-4 C-terminal variants exist in the a or b version depending on the presence or absence of the domain encoded by exon 5. Epilepsy was induced in rats by unilateral intra-amygdalar injection of kainate and animals were killed from 6 h to 4 months following the injection. Increased NR1 mRNA levels were observed during status epilepticus (6-24 h after the injection), both psilateral and contralateral, while a second wave of NMDAR1 mRNA increase occurred in chronic epileptic animals, between 21 days and 4 months following kainate injection. Our data show: (i) a permanent increase of the NR1-2a and NR1-2b mRNA species (containing exon 22) in all hippocampal fields, both ipsilateral and contralateral, and (ii) an increase of the NR1-3 (a and b) mRNAs (containing exon 21) in the ipsilateral CA1, and NR1-3a mRNA in the ipsilateral dentate gyrus. No long-term changes were observed for the NR1-1 and NR14 splice variants. In the ipsilateral CA3 area a globally decreased mRNA expression was associated with neuronal loss. A possible contribution to the maintenance of the epileptic state by an increased expression of NMDA receptors is discussed.     

Developmental changes in NMDA receptor glycine affinity and ifenprodil sensitivity reveal three distinct populations of NMDA receptors in individual rat cortical neurons

Previous work with recombinant receptors has shown that the identity of the NMDA NR2 subunit influences receptor affinity for both glutamate and glycine. We have investigated the developmental change in NMDA receptor affinity for both glutamate and glycine in acutely dissociated parietal cortex neurons of the rat, together with the expression during ontogeny of NR2A and NR2B mRNA and protein. Whereas there is little change in NMDA receptor glutamate affinity with age, a population of NMDA receptors emerges in 14- and 28-d-old animals with a markedly reduced affinity for glycine (mKD = approximately 800 nM) and a reduced sensitivity to the NR2B subunit-selective NMDA antagonist ifenprodil. These changes are paralleled by a developmental increase in the expression of NR2A. Thus, in mature animals a population of NMDA receptors appears with a lower affinity for glycine that might not be saturated under normal physiological conditions. Ifenprodil (10 microM) inhibits virtually all of the NMDA receptor-evoked current in very young neurons that contain a single population of receptors exhibiting a high affinity for glycine (mKD = approximately 20 nM). In older neurons, which contain NMDA receptors with both high and low affinities for glycine, ifenprodil (10 microM) inhibits both the high-affinity population and a significant proportion of the low-affinity component, thus revealing three pharmacologically distinct populations of NMDA receptors in single neurons. Moreover, these observations suggest that ifenprodil might bind with high affinity to NMDA receptors containing both NR2A and NR2B subunits as well as those containing only NR2B.     

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.     

An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.