Characterization of the NR1, NR2A, and NR2C receptor proteins.

N-Methyl-D-aspartate (NMDA) receptor subunits were characterized with seven polyclonal antibodies. The antibodies were directed against NR1-A, NR2A-N1, and NR2C-N1, representing N-terminal sequences of the NR1, NR2A, and NR2C subunits, and against NR1-E, NR2A-C1, and NR2C-C1, derived from C-terminal sequences of these subunits. The anti-NR1-D antibody was raised against the putative internal loop of NR1. A size of 118 kDa was found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis for NR1 (from rat brain) detected by anti-NR1-D and -NR1-E, but not anti-NR1-A. With the anti-NR1-A antibody, a 125-kDa protein was discovered that may represent a glutamate receptor not yet characterized. NR2A and NR2C were identified as proteins with sizes of 175 and 140 kDa, respectively. Enzymatic N-deglycosylation generated a 97-kDa protein from NR1, a 105-kDa protein from the 125-kDa protein, a 162-kDa protein from NR2A, and a 127-kDa protein from NR2C. In contrast to the deglycosylation product of the NR2A, the 97- and 127-kDa proteins derived from NR1 and NR2C, respectively, were found significantly smaller than the molecular masses of 103 and 141 kDa, respectively, predicted on the basis of DNA data. These products may represent truncated proteins. The tissue content of the NR1 and NR2A was high in bovine hippocampus and cortex but lower in the cerebellum. In contrast, NR2C was solely found in the cerebellum. The 125-kDa protein was highest in the cerebellum and cortex.     

Mass spectrometrical identification of hippocampal NMDA receptor subunits NR1, NR2A-D and five novel phosphorylation sites on NR2A and NR2B

The NMDA receptor (NMDA-R) is a key element in neural transmission and mediating a vast variety of physiological and pathological processes in the nervous system. It is well-known that phosphorylation is required for functioning of the NMDA-R, and we therefore decided to study this post-translational modification in subunits NR1 and NR2A-D. Immunoprecipitation with an antibody against NR1 was carried out from rat hippocampi and SDS-PAGEs were run. Bands were punched, destained, and digested with trypsin and chymotrypsin and peptides were identified by nano-LC-ESI-MS/MS using an ion trap (HCT). Proteins were identified using specific software. Phosphorylations were verified by phosphatase treatment and reanalysis by mass spectrometry. The NMDA-R subunits NR1 and 2A-D were identified. On NR2A, a novel phosphorylation site was observed at S511, and on NR2B, four novel phosphorylation sites were revealed at S886, S917, S1303, and S1323 by mass spectrometry and verified by phosphatase treatment with mass spectrometrical reanalysis. A series of NMDA-R phosphorylations have been reported and these serve different functions as receptor activation, localization, and protein-protein interactions. Herein, findings of novel phosphorylation sites are extending knowledge on chemical characterization of the NMDA-R and warrant studying function of site-specific receptor phosphorylation in health and disease.     

NR1, NR2A and NR2C subunits expression after cervical spinal cord transplant and section in dogs

This paper served to evaluate the expression levels of subunits NR1, NR2A and NR2C which are implicated in neuronal plasticity events. A 50% (right half) 4 mm longitudinal resection of the spinal cord was done at the C5-C6 level with preservation of the anterior spinal artery. This was effected in a dog model after either a homologous transplant or a pure spinal cord section. In this study we used two groups of dogs with four individuals each, as well as a control group. The transplant group (n=4) was analyzed at days 3 and 28 post surgery. The section group (n=4) was also analyzed at days 3 and 28 post op. All three groups (transplant, section and control) were evaluated as to the subunit expression in each of the segments corresponding to the transplanted or sectioned sites, the site contralateral to the transplanted or sectioned sites at levels half a centimeter both proximal and distal to the site of transplant and section. The results showed a variety of changes in each of the subunits depending on the group, the segment and the time of evaluation (acute versus chronic). This could be closely related to mechanisms which participate in regeneration and functional recuperation.     

NMDA Receptor Heterogeneity During Postnatal Development of the Rat Brain: Differential Expression of the NR2A, NR2B, and NR2C Subunit Proteins

Abstract: Changes in the expression of the NMDA receptor subunits (NRs) NR2A, 2B, and 2C were investigated in histo blots of the developing rat brain with subunit-specific antisera. At birth, the NR2B subunit was detected almost ubiquitously, the NR2A subunit staining was faint and restricted to the hippocampus, cerebral cortex, and striatum, and no NR2C subunit immunoreactivity was detected. During the first 3 postnatal weeks, the NR2B subunit became confined to forebrain structures, whereas the NR2A immunoreactivity became abundantly expressed throughout the brain. The NR2C immunoreactivity emerged 5 days after birth in the olfactory bulb, thalamus, and vestibular nuclei and became very intense after 10 days in cerebellar granule cells, its primary site of expression in adulthood. After 3 weeks, NR2A and NR2B immunoreactivity decreased to adult levels, whereas NR2C immunoreactivity remained unchanged. The patterns of distribution of the subunit proteins were in agreement with those of their corresponding mRNAs, as monitored by in situ hybridization histochemistry, although the mRNA translation appeared to be delayed by several days in certain areas. Our results reveal a progressive increase in the heterogeneity of NMDA receptors due to the comparably late onset of NR2A and NR2C subunit expression and by the area-specific rearrangement of NR2B subunit expression following birth.     

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

Abstract: 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.     

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.     

Molecular level activation insights from a NR2A/NR2B agonist

N-methyl D-aspartate receptors (NMDARs), a subclass of glutamate receptors have broad actions in neural transmission for major brain functions. Overactivation of NMDARs leading to “excitotoxicity” is the underlying mechanism of neuronal death in a number of neurological diseases, especially stroke. Much research effort has been directed toward developing pharmacological agents to modulate NMDAR actions for treating neurological diseases, in particular stroke. Here, we report that Alliin, a sulfoxide in fresh garlic, exhibits affinity toward NR2A as well as NR2B receptors based on virtual screening. Biological activities of Alliin on these two receptors were confirmed in electrophysiological studies. Ligand-binding site closure, a structural change precluding ion channel opening, was observed with Alliin during 100?ns molecular dynamics simulation. Alliin interactions with NR2A and NR2B suggest that residues E/A413, H485, T690, and Y730 may play important roles in the conformation shift. Activation of NR2A and NR2B by Alliin can be differentiated from that caused by glutamate, the endogenous neurotransmitter. These characteristic molecular features in NR2A and NR2B activation provide insight into structural requirements for future development of novel drugs with selective interaction with NR2A and NR2B for treating neurological diseases, particularly stroke.     

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.     

Triheteromeric NR1/NR2A/NR2B receptors constitute the major N-methyl-D-aspartate receptor population in adult hippocampal synapses

NMDA receptors (NMDARs), fundamental to learning and memory and implicated in certain neurological disorders, are heterotetrameric complexes composed of two NR1 and two NR2 subunits. The function of synaptic NMDARs in postnatal principal forebrain neurons is typically attributed to diheteromeric NR1/NR2A and NR1/NR2B receptors, despite compelling evidence for triheteromeric NR1/NR2A/NR2B receptors. In synapses, the properties of triheteromeric NMDARs could thus far not be distinguished from those of mixtures of diheteromeric NMDARs. To find a signature of NR1/NR2A/NR2B receptors, we have employed two gene-targeted mouse lines, expressing either NR1/NR2A or NR1/NR2B receptors without NR1/NR2A/NR2B receptors, and compared their synaptic properties with those of wild type. In acute hippocampal slices of mutants older than 4 weeks we found a distinct voltage dependence of NMDA R-mediated excitatory postsynaptic current (NMDA EPSC) decay time for the two diheteromeric NMDARs. In wild-type mice, NMDA EPSCs unveiled the NR1/NR2A characteristic for this voltage-dependent deactivation exclusively, indicating that the contribution of NR1/NR2B receptors to evoked NMDA EPSCs is negligible in adult CA3-to-CA1 synapses. The presence of NR1/NR2A/NR2B receptors was obvious from properties that could not be explained by a mixture of diheteromeric NR1/NR2A and NR1/NR2B receptors or by the presence of NR1/NR2A receptors alone. The decay time for NMDA EPSCs in wild type was slower than that for NR1/NR2A receptors, and the sensitivity of NMDA EPSCs to NR2B-directed NMDAR antagonists was 50%. Thus, NR2B is prominent in adult hippocampal synapses as an integral part of NR1/NR2A/NR2B receptors.     

Formation of NR1/NR2 and NR1/NR3 heterodimers constitutes the initial step in N-methyl-D-aspartate receptor assembly

N-Methyl-D-aspartate (NMDA) receptors are tetrameric protein complexes composed of the glycine-binding NR1 subunit with a glutamate-binding NR2 and/or glycine-binding NR3 subunit. Tri-heteromeric receptors containing NR1, NR2, and NR3 subunits reconstitute channels, which differ strikingly in many properties from the respective glycine- and glutamate-gated NR1/NR2 complexes and the NR1/NR3 receptors gated by glycine alone. Therefore, an accurate oligomerization process of the different subunits has to assure proper NMDA receptor assembly, which has been assumed to occur via the oligomerization of homodimers. Indeed, using fluorescence resonance energy transfer analysis of differentially fluorescence-tagged subunits and blue native polyacrylamide gel electrophoresis after metabolic labeling and affinity purification revealed that the NR1 subunit is capable of forming homo-oligomeric aggregates. In contrast, both the NR2 and the NR3 subunits formed homo- and hetero-oligomers only in the presence of the NR1 subunit indicating differential roles of the subunits in NMDA receptor assembly. However, co-expression of the NR3A subunit with an N-terminal domain-deleted NR1 subunit (NR1(DeltaNTD)) abrogating NR1 homo-oligomerization did not affect NR1/NR3A receptor stoichiometry or function. Hence, homo-oligomerization of the NR1 subunit is not essential for proper NR1/NR3 receptor assembly. Because identical results were obtained for NR1(DeltaNTD)/NR2 NMDA receptors (Madry, C., Mesic, I., Betz, H., and Laube, B. (2007) Mol. Pharmacol., 72, 1535-1544) and NR1-containing hetero-oligomers are readily formed, we assume that heterodimerization of the NR1 with an NR3 or NR2 subunit, which is followed by the subsequent association of two heterodimers, is the key step in determining proper NMDA receptor subunit assembly and stoichiometry.     

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.     

Appropriate NR1-NR1 disulfide-linked homodimer formation is requisite for efficient expression of functional, cell surface N-methyl-D-aspartate NR1/NR2 receptors

A c-Myc epitope-tagged N-methyl-D-aspartate receptor NR1-2a subunit was generated, NR1-2a(c-Myc), where the tag was inserted after amino acid 81. NR1-2a(c-Myc) /NR2A receptors when expressed in mammalian cells are not trafficked to the cell surface nor do they yield cell cytotoxicity post-transfection. NR1-2a(c-Myc) was, however, shown to assemble with NR2A subunits by immunoprecipitation and [(3)H]MK801 radioligand binding assays. Immunoblots of cells co-transfected with wild-type NR1-2a/NR2A subunits yielded two NR1-2a immunoreactive species with molecular masses of 115 and 226 kDa. Two-dimensional electrophoresis under non-reducing and reducing conditions revealed that the 226-kDa band contained disulfide-linked NR1-2a subunits. Only the 115-kDa NR1-2a species was detected for NR1-2a(c-Myc)/NR2A. The c-Myc epitope is inserted adjacent to cysteine 79 of the NR1-2a subunit; therefore, it is possible that the tag may prevent the formation of NR1 disulfide bridges. A series of cysteine --> alanine NR1-2a mutants was generated, and the NR1-2a mutants were co-expressed with NR2A or NR2B subunits in mammalian cells and characterized with respect to cell surface expression, cell cytotoxicity post-transfection, co-association by immunoprecipitation, and immunoblotting following SDS-PAGE under both reducing and non-reducing conditions. When co-expressed with NR2A in mammalian cells, NR1-2a(C79A)/NR2A displayed similar properties to NR1-2a(c-Myc)/NR2A in that the 226-kDa NR1 immunoreactive species was not detectable, and trafficking to the cell surface was impaired compared with wild-type NR1/NR2 receptors. These results provide the first biochemical evidence for the formation of NR1-NR1 intersubunit disulfide-linked homodimers involving cysteine 79. They suggest that disulfide bridging and structural integrity within the NR1 N-terminal domain is requisite for cell surface N-methyl-D-aspartate receptor expression.     

NR2A and NR2B subunit containing NMDA receptors differentially regulate striatal output pathways

Triple probe microdialysis was employed to investigate whether striatal NR2A and NR2B subunit containing NMDA receptors regulate the activity of striato-pallidal and striato-nigral projection neurons. Probes were implanted in the striatum, ipsilateral globus pallidus and substantia nigra reticulata. Intrastriatal perfusion with the NR2A subunit selective antagonist ( R )-[( S )-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) reduced pallidal GABA and increased nigral glutamate (GLU) release whereas perfusion with the NR2B subunit selective antagonist ( R -( R *, S *)-α-(4-hydroxyphenyl)-β-methyl-4-(phenylmethyl)-1-piperidinepropanol (Ro 25-6981) reduced nigral GABA and elevated striatal and pallidal GLU release. To confirm that changes in GABA levels were because of blockade of (GLUergic-driven) tonic activity of striatofugal neurons, tetrodotoxin was perfused in the striatum. Tetrodotoxin reduced both pallidal and nigral GABA release without changing GLU levels. To investigate whether striatal NR2A and NR2B subunits were also involved in phasic activation of striatofugal neurons, NVP-AAM077 and Ro 25-6981 were challenged against a NMDA concentration able to evoke GABA release in the three areas. Both antagonists prevented the NMDA-induced striatal GABA release. NVP-AAM077 also prevented the NMDA-induced surge in GABA release in the globus pallidus, whereas Ro 25-6981 attenuated it in the substantia nigra. We conclude that striatal NMDA receptors containing NR2A and NR2B subunits preferentially regulate the striato-pallidal and striato-nigral projection neurons, respectively.     

Fyn tyrosine kinase reduces the ethanol inhibition of recombinant NR1/NR2A but not NR1/NR2B NMDA receptors expressed in HEK 293 cells

NMDA receptors are potentiated by phosphorylation in a subunit- and kinase-specific manner. Both native and recombinant NMDA receptors are inhibited by behaviorally relevant concentrations of ethanol. Whether the phosphorylation state of individual subunits modulates the ethanol sensitivity of these receptors is not known. In this study, the effects of Fyn tyrosine kinase on the ethanol sensitivity of specific recombinant NMDA receptors expressed in HEK 293 cells were investigated. Whole-cell mode patch clamp and ratiometric calcium imaging demonstrated that the degree of ethanol inhibition of NR1/NR2B receptors was unaffected by Fyn tyrosine kinase. In contrast, the inhibition of NR1/NR2A receptors by ethanol (100 mM) was significantly reduced under conditions of enhanced Fyn-mediated tyrosine phosphorylation of the NR2A subunit. This effect was not observed at lower concentrations of ethanol (< or = 50 mM). These results suggest that tyrosine phosphorylation of specific NMDA receptors by Fyn tyrosine kinase may regulate the sensitivity of these receptors to the sedative/hypnotic concentrations of ethanol.     

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.     

Structural rearrangements of NR1/NR2A NMDA receptors during allosteric inhibition

Ionotropic glutamate receptor (iGluR) subunits contain a large N-terminal domain (NTD) that precedes the agonist-binding domain (ABD) and participates in subunit oligomerization. In NMDA receptors (NMDARs), the NTDs of NR2A and NR2B subunits also form binding sites for the endogenous inhibitor Zn(2+) ion. Although these allosteric sites have been characterized in detail, the molecular mechanisms by which the NTDs communicate with the rest of the receptor to promote its inhibition remain unknown. Here, we identify the ABD dimer interface as a major structural determinant that permits coupling between the NTDs and the channel gate. The strength of this interface also controls proton inhibition, another form of allosteric modulation of NMDARs. Conformational rearrangements at the ABD dimer interface thus appear to be a key mechanism conserved in all iGluR subfamilies, but have evolved to fulfill different functions: fast desensitization at AMPA and kainate receptors, allosteric inhibition at NMDARs.     

Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.