NMDA receptor function: subunit composition versus spatial distribution

NMDA receptors (NMDARs) play a pivotal role in the regulation of neuronal communication and synaptic function in the central nervous system. The subunit composition and compartmental localization of NMDARs in neurons affect channel activity and downstream signaling. This review discusses the distinct NMDAR subtypes and their function at synaptic, perisynaptic, and extrasynaptic sites of excitatory and inhibitory neurons. Many neurons express more than one of the modulatory NR2 subunits that participate in the formation of di- and/or triheteromeric channel assemblies (e.g., NR1/NR2A, NR1/NR2B, and/or NR1/NR2A/NR2B). Depending on the subunit composition and presence or absence of intracellular binding partners along the postsynaptic membrane, these NMDAR subtypes are allocated to distinct synaptic inputs converging onto a neuron or are distributed differentially among synaptic or extrasynaptic sites. These sites can carry NR2A and NR2B subunits, supporting the hypothesis that the spatial distribution of scaffolding and signaling complexes critically determines the full spectrum of NMDAR signaling.The author thanks the Deutsche Forschungsgemeinschaft for financial support (Ko 1064/5).     

Involvement of synaptic NR2B-containing NMDA receptors in long-term depression induction in the young rat visual cortex in vitro

Activation of N-methyl-D-aspartate receptors (NMDARs) has been implicated in various forms of synaptic plasticity depending on the receptor subtypes involved. However, the contribution of NR2A and NR2B subunits in the induction of long-term depression (LTD) of excitatory postsynaptic currents (EPSCs) in layer II/III pyramidal neurons of the young rat visual cortex remains unclear. The present study used whole-cell patch-clamp recordings in vitro to investigate the role of NR2A- and NR2B-containing NMDARs in the induction of LTD in visual cortical slices from 12- to 15-day old rats. We found that LTD was readily induced in layer II/III pyramidal neurons of the rat visual cortex with 10-min 1-Hz stimulation paired with postsynaptic depolarization. D-APV, a selective NMDAR antagonist, blocked the induction of LTD. Moreover, the selective NR2B-containing NMDAR antagonists (Ro 25-6981 and ifenprodil) also prevented the induction of LTD. However, Zn2+, a voltage-independent NR2A-containing NMDAR antagonist, displayed no influence on the induction of LTD. These results suggest that the induction of LTD in layer II/III pyramidal neurons of the young rat visual cortex is NMDAR-dependent and requires NR2B-containing NMDARs, not NR2A-containing NMDARs.     

Control of excitatory synaptic transmission by C-terminal Src kinase

The induction of long-term potentiation at CA3-CA1 synapses is caused by an N-methyl-d-aspartate (NMDA) receptordependent accumulation of intracellular Ca(2+), followed by Src family kinase activation and a positive feedback enhancement of NMDA receptors (NMDARs). Nevertheless, the amplitude of baseline transmission remains remarkably constant even though low frequency stimulation is also associated with an NMDAR-dependent influx of Ca(2+) into dendritic spines. We show here that an interaction between C-terminal Src kinase (Csk) and NMDARs controls the Src-dependent regulation of NMDAR activity. Csk associates with the NMDAR signaling complex in the adult brain, inhibiting the Src-dependent potentiation of NMDARs in CA1 neurons and attenuating the Src-dependent induction of long-term potentiation. Csk associates directly with Src-phosphorylated NR2 subunits in vitro. An inhibitory antibody for Csk disrupts this physical association, potentiates NMDAR mediated excitatory postsynaptic currents, and induces long-term potentiation at CA3-CA1 synapses. Thus, Csk serves to maintain the constancy of baseline excitatory synaptic transmission by inhibiting Src kinase-dependent synaptic plasticity in the hippocampus.     

Spatial memory formation induces recruitment of NMDA receptor and PSD-95 to synaptic lipid rafts

NMDA receptors (NMDARs) activation in the hippocampus and insular cortex is necessary for spatial memory formation. Recent studies suggest that localization of NMDARs to lipid rafts enhance their signalization, since the kinases that phosphorylate its subunits are present in larger proportion in lipid raft membrane microdomains. We sought to determine the possibility that NMDAR translocation to synaptic lipid rafts occurs during plasticity processes such as memory formation. Our results show that water maze training induces a rapid recruitment of NMDAR subunits (NR1, NR2A, NR2B) and PSD-95 to synaptic lipid rafts and decrease in the post-synaptic density plus an increase of NR2B phosphorylation at tyrosine 1472 in the rat insular cortex. In the hippocampus, spatial training induces selective translocation of NR1 and NR2A subunits to lipid rafts. These results suggest that NMDARs translocate from the soluble fraction of post-synaptic membrane (non-raft PSD) to synaptic lipid raft during spatial memory formation. The recruitment of NMDA receptors and other proteins to lipid rafts could be an important mechanism for increasing the efficiency of synaptic transmission during synaptic plasticity process.     

Subunit-specific modulation of [(3)H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [(3)H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [(3)H]MK-801 binding with increased potency in membranes from the NR2A(-/-) mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [(3)H]MK-801 binding, all showing half maximal inhibitory concentrations ~20 μM, and with significant effects occurring at or above 1 μM. With membranes from D1R(-/-) mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.     

Protein kinase CK2 in postsynaptic densities: phosphorylation of PSD-95/SAP90 and NMDA receptor regulation

Protein kinase CK2 (CK2) is highly expressed in rat forebrain where its function is not well understood. Subcellular distribution studies showed that the catalytic subunit of CK2 (CK2alpha) was enriched in postsynaptic densities (PSDs) by 68%. We studied the putative role of CK2 activity on N-methyl-D-aspartate receptor (NMDAR) function using isolated, patch-clamped PSDs in the presence of 2 mM extracellular Mg(2+). The usual activation by phosphorylation of the NMDARs in the presence of ATP was inhibited by the selective CK2 inhibitor 5,6-dichloro-1-beta-ribofuranosyl benzimidazole (DRB). This inhibition was voltage-dependent, i.e., 100% at positive membrane potentials, while at negative potentials, inhibition was incomplete. Endogenous CK2 substrates were characterized by their ability to use GTP as a phosphoryl donor and susceptibility to inhibition by DRB. Immunoprecipitation assays and 2D gels indicated that PSD-95/SAP90, the NMDAR scaffolding protein, was a CK2 substrate, while the NR2A/B and NR1 NMDAR subunits were not. These results suggest that postsynaptic NMDAR regulation by CK2 is mediated by indirect mechanisms possibly involving PSD-95/SAP90.     

Ethanol alters trafficking and functional N-methyl-D-aspartate receptor NR2 subunit ratio via H-Ras

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity and is one of the main targets for alcohol (ethanol) in the brain. Trafficking of the NMDAR is emerging as a key regulatory mechanism that underlies channel activity and synaptic plasticity. Here we show that exposure of hippocampal neurons to ethanol increases the internalization of the NR2A but not NR2B subunit of the NMDAR via the endocytic pathway. We further observed that ethanol exposure results in NR2A endocytosis through the activation of H-Ras and the inhibition of the tyrosine kinase Src. Importantly, ethanol treatment alters functional subunit composition from NR2A/NR2B- to mainly NR2B-containing NMDARs. Our results suggest that addictive drugs such as ethanol alter NMDAR trafficking and subunit composition. This may be an important mechanism by which ethanol exerts its effects on NMDARs to produce alcohol-induced aberrant plasticity.     

Regulation of calcium/calmodulin-dependent protein kinase II docking to N-methyl-D-aspartate receptors by calcium/calmodulin and alpha-actinin

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.     

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum

We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS). We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits. In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.     

The NMDA Receptor NR1 Subunit is Critically Involved in the Regulation of NMDA Receptor Activity by C-terminal Src kinase (Csk)

Previous studies have shown that Csk plays critical roles in the regulation of neural development, differentiation and glutamate-mediated synaptic plasticity. It has been found that Csk associates with the NR2A and 2B subunits of N-methyl-D-aspartate receptors (NMDARs) in a Src activity-dependent manner and serves as an intrinsic mechanism to provide a “brake” on the induction of long-term synaptic potentiation (LTP) mediated by NMDARs. In contrast to the NR2A and 2B subunits, no apparent tyrosine phosphorylation is found in the NR1 subunit of NMDARs. Here, we report that Csk can also associate with the NR1 subunit in a Src activity-dependent manner. The truncation of the NR1 subunit C-tail which contains only one tyrosine (Y837) significantly reduced the Csk association with the NR1-1a/NR2A receptor complex. Furthermore, we found that either the truncation of NR2A C-tail at aa 857 or the mutation of Y837 in the NR1-1a subunit to phenylalanine blocked the inhibition of NR1-1a/NR2A receptors induced by intracellular application of Csk. Thus, both the NR1 and NR2 subunits are required for the regulation of NMDAR activity by Csk.     

Mind bomb-2 is an E3 ligase that ubiquitinates the N-methyl-D-aspartate receptor NR2B subunit in a phosphorylation-dependent manner

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination.     

Obligatory role of NR2A for metaplasticity in visual cortex

Light deprivation lowers the threshold for long-term depression (LTD) and long-term potentiation (LTP) in visual cortex by a process termed metaplasticity, but the mechanism is unknown. The decreased LTD/P threshold correlates with a decrease in the ratio of NR2A to NR2B subunits of cortical NMDA receptors (NMDARs) and a slowing of NMDAR-mediated excitatory postsynaptic currents (EPSCs). However, whether and how changes in NR2 subunit expression contribute to LTD and LTP have been controversial. In the present study, we used an NR2A knockout (KO) mouse to examine the role of this subunit in the experience-dependent modulation of NMDAR properties, LTD, and LTP. We found that deletion of NR2A abrogates the effects of visual experience on NMDAR EPSCs and prevents metaplasticity of LTP and LTD. These data support the hypothesis that experience-dependent changes in NR2A/B are functionally significant and yield a mechanism for an adjustable synaptic modification threshold in visual cortex.     

The synaptic localization of NR2B-containing NMDA receptors is controlled by interactions with PDZ proteins and AP-2

The NMDA receptor (NMDAR) is a component of excitatory synapses and a key participant in synaptic plasticity. We investigated the role of two domains in the C terminus of the NR2B subunit--the PDZ binding domain and the clathrin adaptor protein (AP-2) binding motif--in the synaptic localization of NMDA receptors. NR2B subunits lacking functional PDZ binding are excluded from the synapse. Mutations in the AP-2 binding motif, YEKL, significantly increase the number of synaptic receptors and allow the synaptic localization of NR2B subunits lacking PDZ binding. Peptides corresponding to YEKL increase the synaptic response within minutes. In contrast, the NR2A subunit localizes to the synapse in the absence of PDZ binding and is not altered by mutations in its motif corresponding to YEKL of NR2B. This study identifies a dynamic regulation of synaptic NR2B-containing NMDARs through PDZ protein-mediated stabilization and AP-2-mediated internalization that is modulated by phosphorylation by Fyn kinase.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.     

NR2B-containing NMDA receptors promote neural progenitor cell proliferation through CaMKIV/CREB pathway

Accumulating evidence indicates the involvement of N-methyl-d-aspartate receptors (NMDARs) in regulating neural stem/progenitor cell (NSPC) proliferation. Functional properties of NMDARs can be markedly influenced by incorporating the regulatory subunit NR2B. Here, we aim to analyze the effect of NR2B-containing NMDARs on the proliferation of hippocampal NSPCs and to explore the mechanism responsible for this effect. NSPCs were shown to express NMDAR subunits NR1 and NR2B. The NR2B selective antagonist, Ro 25-6981, prevented the NMDA-induced increase in cell proliferation. Moreover, we demonstrated that the phosphorylation levels of calcium/calmodulin-dependent protein kinase IV (CaMKIV) and cAMP response element binding protein (CREB) were increased by NMDA treatment, whereas Ro 25-6981 decreased them. The role that NR2B-containing NMDARs plays in NSPC proliferation was abolished when CREB phosphorylation was attenuated by CaMKIV silencing. These results suggest that NR2B-containing NMDARs have a positive role in regulating NSPC proliferation, which may be mediated through CaMKIV phosphorylation and subsequent induction of CREB activation.     

Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-( t -butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.     

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.     

NADPH oxidase mediates the oxygen-glucose deprivation/reperfusion-induced increase in the tyrosine phosphorylation of the N-methyl-D-aspartate receptor NR2A subunit in retinoic acid differentiated SH-SY5Y Cells

ABSTRACT: BACKGROUND: Evidence exists that oxidative stress promotes the tyrosine phosphorylation of N-methyl-D-aspartate receptor (NMDAR) subunits during post-ischemic reperfusion of brain tissue. Increased tyrosine phosphorylation of NMDAR NR2A subunits has been reported to potentiate receptor function and exacerbate NMDAR-induced excitotoxicity. Though the effect of ischemia on tyrosine phosphorylation of NMDAR subunits has been well documented, the oxidative stress signaling cascades mediating the enhanced tyrosine phosphorylation of NR2A subunits remain unclear. RESULTS: We report that the reactive oxygen species (ROS) generator NADPH oxidase mediates an oxidative stress-signaling cascade involved in the increased tyrosine phosphorylation of the NR2A subunit in post-ischemic differentiated SH-SY5Y neuroblastoma cells. Inhibition of NADPH oxidase attenuated the increased tyrosine phosphorylation of the NMDAR NR2A subunit, while inhibition of ROS production from mitochondrial or xanthine oxidase sources failed to dampen the post-ischemic increase in tyrosine phosphorylation of the NR2A subunit. Additionally, inhibition of NADPH oxidase blunted the interaction of activated Src Family Kinases (SFKs) with PSD-95 induced by ischemia/reperfusion. Lastly, inhibition of NADPH oxidase also markedly reduced cell death in post-ischemic SH-SY5Y cells stimulated by NMDA. CONCLUSIONS: These data indicate that NADPH oxidase has a key role in facilitating NMDAR NR2A tyrosine phosphorylation via SFK activation during post-ischemic reperfusion.     

Marked attenuation of inflammatory mediator-induced C-fiber sensitization for mechanical and hypotonic stimuli in TRPV4-/- mice

NMDA receptors (NMDARs) are involved in excitatory synaptic transmission and plasticity associated with a variety of brain functions, from memory formation to chronic pain. Subunit-selective antagonists for NMDARs provide powerful tools to dissect NMDAR functions in neuronal activities. Recently developed antagonist for NR2A-containing receptors, NVP-AAM007, triggered debates on its selectivity and involvement of the NMDAR subunits in bi-directional synaptic plasticity. Here, we re-examined the pharmacological properties of NMDARs in the anterior cingulate cortex (ACC) using NVP-AAM007 as well as ifenprodil, a selective antagonist for NR2B-containing NMDARs. By alternating sequence of drug application and examining different concentrations of NVP-AAM007, we found that the presence of NVP-AAM007 did not significantly affect the effect of ifenprodil on NMDAR-mediated EPSCs. These results suggest that NVP-AAM007 shows great preference for NR2A subunit and could be used as a selective antagonist for NR2A-containing NMDARs in the ACC.