Glutamate Binding to the GluN2B Subunit Controls Surface Trafficking of N-Methyl-D-aspartate (NMDA) Receptors

Trafficking of NMDA receptors to the surface of neurons and to synapses is critical for proper brain function and activity-dependent plasticity. Recent evidence suggests that surface trafficking of other ionotropic glutamate receptors requires ligand binding for exit from the endoplasmic reticulum. Here, we show that glutamate binding to GluN2 is required for trafficking of NMDA receptors to the cell surface. We expressed a panel of GluN2B ligand binding mutants in heterologous cells with GluN1 or in rat cultured neurons and found that surface expression correlates with glutamate efficacy. Such a correlation was found even in the presence of dominant negative dynamin to inhibit endocytosis and surface expression correlated with Golgi localization, indicating differences in forward trafficking. Co-expression of wild type GluN2B did not enhance surface expression of the mutants, suggesting that glutamate must bind to both GluN2 subunits in a tetramer and that surface expression is limited by the least avid of the two glutamate binding sites. Surface trafficking of a constitutively closed cleft GluN2B was indistinguishable from that of wild type, suggesting that glutamate concentrations are typically not limiting for forward trafficking. YFP-GluN2B expressed in hippocampal neurons from GluN2B(-/-) mice rescued synaptic accumulation at similar levels to wild type. Under these conditions, surface synaptic accumulation of YFP-GluN2B mutants also correlated with apparent glutamate affinity. Altogether, these results indicate that glutamate controls forward trafficking of NMDA receptors to the cell surface and to synapses and raise the intriguing idea that NMDA receptors may be functional at intracellular sites.     

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca²⁺-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca²⁺-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca²⁺, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca²⁺ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca²⁺ affects CDI, we recorded one-channel Na⁺ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca²⁺ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (P_o). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high P_o GluN2B receptors (GluN1^A652Y/GluN2B). Together, these results show that Ca²⁺ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel P_o. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca²⁺ load during high P_o activation.     

Activated CaMKII Couples GluN2B and Casein Kinase 2 to Control Synaptic NMDA Receptors

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Highlights? Activated CaMKII recruits CK2 to form a trimolecular complex (GluN2B/CaMKII/CK2) ? GluN2B/CaMKII binding controls CK2 phosphorylation of the GluN2B PDZ ligand (S1480) ? Disruption of GluN2B/CaMKII binding increases GluN2B surface expression ? GluN2B/CaMKII binding drives synaptic GluN2B clearance     

Palmitoylation at Two Cysteine Clusters on the C-Terminus of GluN2A and GluN2B Differentially Control Synaptic Targeting of NMDA Receptors

Palmitoylation of NMDARs occurs at two distinct cysteine clusters in the carboxyl-terminus of GluN2A and GluN2B subunits that differentially regulates retention in the Golgi apparatus and surface expression of NMDARs. Mutations of palmitoylatable cysteine residues in the membrane-proximal cluster to non-palmitoylatable serines leads to a reduction in the surface expression of recombinant NMDARs via enhanced internalization of the receptors. Mutations in a cluster of cysteines in the middle of the carboxyl-terminus of GluN2A and GluN2B, leads to an increase in the surface expression of NMDARs via an increase in post-Golgi trafficking. Using a quantitative electrophysiological assay, we investigated whether palmitoylation of GluN2 subunits and the differential regulation of surface expression affect functional synaptic incorporation of NMDARs. We show that a reduction in surface expression due to mutations in the membrane-proximal cluster translates to a reduction in synaptic expression of NMDARs. However, increased surface expression induced by mutations in the cluster of cysteines that regulates post-Golgi trafficking of NMDARs does not increase the synaptic pool of NMDA receptors, indicating that the number of synaptic receptors is tightly regulated.     

Expression of the Hippocampal NMDA Receptor GluN1 Subunit and Its Splicing Isoforms in Schizophrenia: Postmortem Study

There is accumulating evidence that disturbances in N-methyl-d-aspartate receptor (NMDA-R) functioning are associated with the pathogenesis of schizophrenia. To assess actual changes in the expression of the GluN1 subunit and its isoforms, we measured absolute differences in the levels of mRNA/protein for panGluN1 (eight isoforms altogether) as well as the mRNA individual isoforms in the postmortem left/right hippocampus of patients with schizophrenia in comparison with non-psychiatric subjects. There were no significant differences in the panGluN1 subunit mRNA expression, but the absolute left/right differences were much more pronounced in the patients with schizophrenia. Protein levels of the GluN1 subunit in the left hippocampus in male schizophrenic patients were lower than controls. The expression of the NR1-4b isoform was attenuated in the left, whereas the NR1-2b was reduced in the right hippocampus of schizophrenic patients. Isoforms associated with the efficiency of NMDA-induced gene expression and with phosphorylation occurred more commonly in schizophrenic hippocampi. In summary, our study suggests that NMDA-R hypofunction in schizophrenia might be selectively dependent on the dysregulation of GluN1 subunit expression, which exhibits a somewhat different expression in the left/right hippocampus of psychotic patients.     

Deletion of the NMDA Receptor GluN2A Subunit Significantly Decreases Dendritic Growth in Maturing Dentate Granule Neurons

It is known that NMDA receptors can modulate adult hippocampal neurogenesis, but the contribution of specific regulatory GluN2 subunits has been difficult to determine. Here we demonstrate that mice lacking GluN2A (formerly NR2A) do not show altered cell proliferation or neuronal differentiation, but present significant changes in neuronal morphology in dentate granule cells. Specifically, GluN2A deletion significantly decreased total dendritic length and dendritic complexity in DG neurons located in the inner granular zone. Furthermore, the absence of GluN2A also resulted in a localized increase in spine density in the middle molecular layer, a region innervated by the medial perforant path. Interestingly, alterations in dendritic morphology and spine density were never seen in dentate granule cells located in the outer granular zone, a region that has been hypothesized to contain older, more mature, neurons. These results indicate that although the GluN2A subunit is not critical for the cell proliferation and differentiation stages of the neurogenic process, it does appear to play a role in establishing synaptic and dendritic morphology in maturing dentate granule cells localized in the inner granular zone.     

Two N-glycosylation Sites in the GluN1 Subunit Are Essential for Releasing N-methyl-d-aspartate (NMDA) Receptors from the Endoplasmic Reticulum

NMDA receptors (NMDARs) comprise a subclass of neurotransmitter receptors whose surface expression is regulated at multiple levels, including processing in the endoplasmic reticulum (ER), intracellular trafficking via the Golgi apparatus, internalization, recycling, and degradation. With respect to early processing, NMDARs are regulated by the availability of GluN subunits within the ER, the presence of ER retention and export signals, and posttranslational modifications, including phosphorylation and palmitoylation. However, the role of N-glycosylation, one of the most common posttranslational modifications, in regulating NMDAR processing has not been studied in detail. Using biochemistry, confocal and electron microscopy, and electrophysiology in conjunction with a lentivirus-based molecular replacement strategy, we found that NMDARs are released from the ER only when two asparagine residues in the GluN1 subunit (Asn-203 and Asn-368) are N-glycosylated. Although the GluN2A and GluN2B subunits are also N-glycosylated, their N-glycosylation sites do not appear to be essential for surface delivery of NMDARs. Furthermore, we found that removing N-glycans from native NMDARs altered the receptor affinity for glutamate. Our results suggest a novel mechanism by which neurons ensure that postsynaptic membranes contain sufficient numbers of functional NMDARs.     

Phosphorylation of Tyrosine 1070 at the GluN2B Subunit Is Regulated by Synaptic Activity and Critical for Surface Expression of N-Methyl-d-aspartate (NMDA) Receptors

The number and subunit composition of synaptic N-methyl-d-aspartate receptors (NMDARs) play critical roles in synaptic plasticity, learning, and memory and are implicated in neurological disorders. Tyrosine phosphorylation provides a powerful means of regulating NMDAR function, but the underling mechanism remains elusive. In this study we identified a tyrosine site on the GluN2B subunit, Tyr-1070, which was phosphorylated by a proto-oncogene tyrosine-protein (Fyn) kinase and critical for the surface expression of GluN2B-containing NMDARs. The phosphorylation of GluN2B at Tyr-1070 was required for binding of Fyn kinase to GluN2B, which up-regulated the phosphorylation of GluN2B at Tyr-1472. Moreover, our results revealed that the phosphorylation change of GluN2B at Tyr-1070 accompanied the Tyr-1472 phosphorylation and Fyn associated with GluN2B in synaptic plasticity induced by both chemical and contextual fear learning. Taken together, our findings provide a new mechanism for regulating the surface expression of NMDARs with implications for synaptic plasticity.     

Pharmacological Characterization of Heterodimeric NMDA Receptors Composed of NR 1a and 2B Subunits: Differences with Receptors Formed from NR 1a and 2A

Abstract: Pharmacological and molecular biological evidence indicates the existence of multiple types of NMDA receptors within the CNS. We have characterized pharmacological properties of receptors assembled from the combination of NR 1a and NR 2B subunits (NR 1a/2B) expressed in transfected cells using both ¹²⁵I-MK-801 binding assays and electrophysiological measures. Binding of ¹²⁵I-MK-801 to cells transfected with NR 1a/2B is saturable with a K _D of 440 p M . The binding is potently inhibited by ketamine, dextromethorphan, phencyclidine, and MK-801 and is stimulated by low concentrations of magnesium. These properties resemble those of native receptors and receptors produced by NR 1a/2A. However, ¹²⁵I-MK-801 binding to membranes from cells transfected with NR 1a/2B is inhibited with high affinity by ifenprodil and is stimulated by spermidine, unlike receptors assembled from NR 1a/2A. NMDA-induced currents measured in cells transfected with either NR 1a/2A or NR 1a/2B have pharmacological properties that correlate well with the binding studies. Currents in cells transfected with NR 1a/2B are potentiated by spermidine and blocked with high affinity by ifenprodil, whereas currents in cells transfected with NR 1a/2A are not enhanced by spermidine and are weakly inhibited by ifenprodil. These data suggest that pharmacological heterogeneity in native NMDA receptors may be explained by combinations of different subunits.     

Developmental and Regional Expression of NMDA Receptor Subtypes Containing the NR2D Subunit in Rat Brain

Abstract: The regional and developmental expression of NMDA receptors containing the NR2D subunit was analyzed on the level of the subunit mRNA and protein in rat brain. RNase protection experiments indicated that among two proposed splice variants of the NR2D subunit, only the NR2D-2 subunit is expressed. The regional distribution of the NR2D subunit protein was visualized with a newly developed NR2D-2 subunit-specific antiserum on brain sections using the histoblot technique. In adult brain, NR2D immunoreactivity was mainly restricted to diencephalic, mesencephalic, and brainstem structures. During postnatal development, the NR2D subunit was detected transiently in certain regions, such as the ventro-basal complex of the thalamus, hippocampus, inferior colliculus, and brainstem reticular formation, suggesting that NR2D subunit-containing receptors play a role in these brain areas only during development. The level of NR2D subunit mRNA and protein decreased during late postnatal development. However, significant levels of NR2D subunit mRNA and protein were present in adulthood, in particular, in the globus pallidus, thalamus, subthalamic nuclei, and superior colliculus. These results indicate a functional relevance for NMDA receptors containing the NR2D subunit in the developing and adult brain, although its expression in the adult brain is less prominent and restricted to a few brain areas.     

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.     

Distinct modes of AMPA receptor suppression at developing synapses by GluN2A and GluN2B: single-cell NMDA receptor subunit deletion in vivo

During development there is an activity-dependent switch in synaptic N-Methyl-D-aspartate (NMDA) receptor subunit composition from predominantly GluN2B to GluN2A, though the precise role of this?switch remains unknown. By deleting GluN2 subunits in single neurons during synaptogenesis, we find that both GluN2B and GluN2A suppress AMPA receptor expression, albeit by distinct means. Similar to GluN1, GluN2B deletion increases the number of functional synapses, while GluN2A deletion increases the strength of unitary connections without affecting the number of functional synapses. We propose a model of excitatory synapse maturation in which baseline activation of GluN2B-containing receptors prevents premature synapse maturation until correlated activity allows induction of functional synapses. This activity also triggers the switch to GluN2A, which dampens further potentiation. Furthermore, we analyze the subunit composition of synaptic NMDA receptors in CA1 pyramidal cells, provide electrophysiological evidence for?a large population of synaptic triheteromeric receptors, and estimate the subunit-dependent open probability.     

Nucleotides and Phosphorylation Bi-directionally Modulate Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) Binding to the N-Methyl-d-aspartate (NMDA) Receptor Subunit GluN2B

The Ca²⁺/calmodulin (CaM)-dependent protein kinase II (CaMKII) and the NMDA-type glutamate receptor are key regulators of synaptic plasticity underlying learning and memory. Direct binding of CaMKII to the NMDA receptor subunit GluN2B (formerly known as NR2B) (i) is induced by Ca²⁺/CaM but outlasts this initial Ca²⁺-stimulus, (ii) mediates CaMKII translocation to synapses, and (iii) regulates synaptic strength. CaMKII binds to GluN2B around S1303, the major CaMKII phosphorylation site on GluN2B. We show here that a phospho-mimetic S1303D mutation inhibited CaM-induced CaMKII binding to GluN2B in vitro, presenting a conundrum how binding can occur within cells, where high ATP concentration should promote S1303 phosphorylation. Surprisingly, addition of ATP actually enhanced the binding. Mutational analysis revealed that this positive net effect was caused by four modulatory effects of ATP, two positive (direct nucleotide binding and CaMKII T286 autophosphorylation) and two negative (GluN2B S1303 phosphorylation and CaMKII T305/6 autophosphorylation). Imaging showed positive regulation by nucleotide binding also within transfected HEK cells and neurons. In fact, nucleotide binding was a requirement for efficient CaMKII interaction with GluN2B in cells, while T286 autophosphorylation was not. Kinetic considerations support a model in which positive regulation by nucleotide binding and T286 autophosphorylation occurs faster than negative modulation by GluN2B S1303 and CaMKII T305/6 phosphorylation, allowing efficient CaMKII binding to GluN2B despite the inhibitory effects of the two slower reactions.     

Dimeric Pentapeptide and Tetrapeptide Enkephalins: New Tools for the Study of Delta Opioid Receptors

Abstract

When a dimeric ligand can react bivalently, one would expect an increase in affinity, selectivity, and possibly biological activity. On this premise, we have synthesized and characterized two series of dimers, viz.: Dimeric Pentapeptide Enkephalin (DPE_n = (H-Tyr-D-Ala-Gly-Phe-Leu-NH-)₂ · (CH₂)_n, and Dimeric Tetrapeptide Enkephalin (DTE_n) = (H-Tyr-D-Ala-Gly-Phe-NH)₂ · (CH₂)_n, with n = 2, 4, …, 12. These dimers display affinity, activity, and δ/μ selectivity which vary systematically with chain length (n). DPE₂ shows a seven-fold increase in affinity for the δ receptor of whole brain and NG108-15 cells, relative to monomer, while its activity for the μ receptor is similar to enkephalin monomers. DTE₁₂ shows a dramatic increase in δ selectivity relative to its monomer. The association rate constant for ³H-DPE₂ is increased two-fold and its dissociation rate constant is significantly reduced, relative to monomer. DPE₂ shows a loss of affinity in the presence of Na⁺ or Mn⁺⁺, while GTP unexpectedly increases its affinity under some conditions. DPE₂ shows equal potency with the agonist [D-Ala², D-Leu⁵] enkephalin in assays measuring their inhibitory effect on prostaglandin E₁-stimulated cAMP production by NG108-15 cells. DPE₂ is very potent in the mouse vas deferens assay; DTE₁₂ shows substantially less activity. These results suggest that δ opiate receptors may be closely clustered in the cell membrane, and provide new approaches to the development of δ and μ receptor ligands.     

A critical role for GluN2B-containing NMDA receptors in cortical development and function

The subunit composition of N-methyl D-aspartate receptors (NMDARs) is tightly regulated during cortical development. NMDARs are initially dominated by GluN2B (NR2B), whereas GluN2A (NR2A) incorporation increases after birth. The function of GluN2B-containing NMDARs during development, however, is incompletely understood. We generated a mouse in which we genetically replaced GluN2B with GluN2A (2B→2A). Although this manipulation restored NMDAR-mediated currents at glutamatergic synapses, it did not rescue GluN2B loss of function. Protein translation-dependent homeostatic synaptic plasticity is occluded in the absence of GluN2B, and AMPA receptor contribution is enriched at excitatory cortical synapses. Our experiments indicate that specificity of GluN2B-mediated signaling is due to its unique interaction with the protein effector alpha calcium-calmodulin kinase II and the regulation of the mTOR pathway. Homozygous 2B→2A mice exhibited high rates of lethality, suppressed feeding, and depressed social exploratory behavior. These experiments indicate that GluN2B-containing NMDARs activate unique cellular processes that cannot be rescued by replacement with GluN2A.     

Gating mechanism and a modulatory niche of human GluN1-GluN2A NMDA receptors

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