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1.  Ethanol Inhibition of Recombinant NMDA Receptors Is Not Altered by Co-Expression of CaMKII-α or CaMKII-β 
Alcohol (Fayetteville, N.Y.)  2008;42(5):425-432.
Previous studies have shown that the N-methyl-D-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. NMDA receptors are glutamate-activated ion channels that are highly expressed in neurons. They are activated during periods of significant glutamatergic synaptic activity and are an important source of the signaling molecule calcium in the post-synaptic spine. Alterations in the function of NMDA receptors by drugs or disease are associated with deficits in motor, sensory and cognitive processes of the brain. Acutely, ethanol inhibits ion flow through NMDA receptors while sustained exposure to ethanol can induce compensatory changes in the density and localization of the receptor. Defining factors that govern the acute ethanol sensitivity of NMDA receptors is an important step in how an individual responds to ethanol. In the present study, we investigated the effect of calcium-calmodulin dependent protein kinase II (CaMKII) on the ethanol sensitivity of recombinant NMDA receptors. CaMKII is a major constituent of the post-synaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells (HEK 293) along with CaMKII-α or CaMKII-β tagged with the green fluorescent protein (GFP). Whole cell currents were elicited by brief exposures to glutamate and were measured using patchclamp electrophysiology. Neither CaMKII-α or CaMKII-β had any significant effect on the ethanol inhibition of NR1/2A or NR1/2B receptors. Ethanol inhibition was also unaltered by deletion of CaMKII binding domains in NR1 or NR2 subunits or by phospho-site mutants that mimic or occlude CaMKII phosphorylation. Chronic treatment of cortical neurons with ethanol had no significant effect on the expression of CaMKII-α or CaMKII-β. The results of this study suggest that CaMKII is not involved in regulating the acute ethanol sensitivity of NMDA receptors.
doi:10.1016/j.alcohol.2008.04.007
PMCID: PMC2629600  PMID: 18562151
electrophysiology; alcohol; ion channel; kinase; phosphorylation
2.  Enhanced Ethanol Inhibition of Recombinant NMDA Receptors by Magnesium: Role of NR3 Subunits 
Background
The effects of ethanol on brain function are thought to be due in part to alterations in the activity of ion channels that regulate synaptic activity. Results from previous studies from this lab and others have shown that ethanol inhibits the function of the N-methyl-D-aspartate (NMDA) receptors, a calcium-permeable ion channel activated by the neurotransmitter glutamate. Factors that alter the acute sensitivity of NMDA receptors to ethanol may be critical in determining how neurons and neuronal networks respond to the presence of ethanol. In this study, we have examined the effect of physiologically relevant concentrations of magnesium on the ethanol sensitivity of recombinant NMDA receptors and how ethanol inhibition under these conditions is influenced by the NR3A subunit.
Methods
Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney (HEK) 293 cells. Whole-cell patch-clamp electrophysiology was used to measure currents induced by rapid application of glutamate in the absence and presence of ethanol.
Results
In magnesium-free recording solution, ethanol inhibited glutamate-mediated currents in cells transfected with NMDA receptor subunits. The magnitude of ethanol inhibition was significantly enhanced when recordings were carried out in media containing 1 mM magnesium. This effect was reversible and required magnesium-sensitive receptors. Magnesium did not enhance ethanol inhibition of glycine-activated NR1/NR3A/NR3B receptors. However, NR3A co-expression prevented the enhancement of ethanol's inhibitory effect on receptors composed of NR2A but not NR2B subunits.
Conclusions
These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors
doi:10.1111/j.1530-0277.2008.00667.x
PMCID: PMC3590455  PMID: 18445116
electrophysiology; addiction; glutamate; HEK cells
3.  Dual regulation by ethanol of the inhibitory effects of ketamine on spinal NMDA-induced pressor responses in rats 
Background
Acute exposure of ethanol (alcohol) inhibits NMDA receptor function. Our previous study showed that acute ethanol inhibited the pressor responses induced by NMDA applied intrathecally; however, prolonged ethanol exposure may increase the levels of phosphorylated NMDA receptor subunits leading to changes in ethanol inhibitory potency on NMDA-induced responses. The present study was carried out to examine whether acute ethanol exposure influences the effects of ketamine, a noncompetitive NMDA receptor antagonist, on spinal NMDA-induced pressor responses.
Methods
The blood pressure responses induced by intrathecal injection of NMDA were recorded in urethane-anesthetized rats weighing 250-275 g. The levels of several phosphorylated residues on NMDA receptor GluN1 subunits were determined by western blot analysis.
Results
Intravenous injection of ethanol or ketamine inhibited spinal NMDA-induced pressor responses in a dose-dependent and reversible manner. Ketamine inhibition of NMDA-induced responses was synergistically potentiated by ethanol when ethanol was applied just before ketamine. However, ketamine inhibition was significantly reduced when applied at 10 min after ethanol administration. Western blot analysis showed that intravenous ethanol increased the levels of phosphoserine 897 on GluN1 subunits (pGluN1-serine 897), selectively phosphorylated by protein kinase A (PKA), in the lateral horn regions of spinal cord at 10 min after administration. Intrathecal administration of cAMPS-Sp, a PKA activator, at doses elevating the levels of pGluN1-serine 897, significantly blocked ketamine inhibition of spinal NMDA-induced responses.
Conclusions
The results suggest that ethanol may differentially regulate ketamine inhibition of spinal NMDA receptor function depending on ethanol exposure time and the resulting changes in the levels of pGluN1-serine 897.
doi:10.1186/1423-0127-19-11
PMCID: PMC3296648  PMID: 22300389
alcohol; ketamine; NMDA receptor; PKA; phosphorylation; sympathetic neuron
4.  Dephosphorylation of GluN2B C-Terminal Tyrosine Residues Does Not Contribute to Acute Ethanol Inhibition of Recombinant NMDA Receptors 
Alcohol (Fayetteville, N.Y.)  2013;47(3):181-186.
N-methyl-D-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are highly expressed by neurons. These receptors are critical for excitatory synaptic signaling and inhibition of NMDA receptors leads to impaired cognition and learning. Ethanol inhibits NMDA currents at concentrations associated with intoxication and this action may underlie some of the behavioral effects of ethanol. Although numerous sites and mechanisms of action have been tested, the manner in which ethanol inhibits NMDA receptors remains unclear. Recent findings in the literature suggest that ethanol, via facilitation of tyrosine phosphatase activity, may dephosphorylate key tyrosine residues in the C-terminus of GluN2B subunits resulting in diminished channel function. To directly test this hypothesis, we engineered GluN2B mutants that contained phenylalanine in place of tyrosine at three different sites and transiently expressed them with the GluN1 subunit in human embryonic kidney (HEK) cells. Whole-cell patch clamp electrophysiology was used to record glutamate-activated currents in the absence and presence of ethanol (10–600 mM). All mutants were functional and did not differ from one another with respect to current amplitude, steady-state to peak ratio, or magnesium block. Analysis of ethanol dose-response curves showed no significant difference in IC50 values between wild-type receptors and Y1252F, Y1336F, Y1472F or triple Y-F mutants. These findings suggest that dephosphorylation of C-terminal tyrosine residues does not account for ethanol inhibition of GluN2B receptors.
doi:10.1016/j.alcohol.2012.12.015
PMCID: PMC3617063  PMID: 23357553
Ethanol; phosphorylation; GluN2B; Electrophysiology
5.  Deletion of the N-terminal Domain (NTD) Alters the Ethanol Inhibition of NMDA Receptors in a Subunit-Dependent Manner 
Alcoholism, clinical and experimental research  2013;37(11):10.1111/acer.12168.
Background
Ethanol inhibition of NMDA receptors is poorly understood due in part to the organizational complexity of the receptor that provides ample locations for sites of action. Among these the N-terminal domain of NMDA receptor subunits contains binding sites for a variety of modulatory agents including zinc, protons and GluN2B selective antagonists such as ifenprodil or Ro-25–6981. Ethanol inhibition of neuronal NMDA receptors expressed in some brain areas has been reported to be occluded by the presence of ifenprodil or similar compounds suggesting that the N-terminal domain may be important in regulating the ethanol sensitivity of NMDA receptors.
Methods
Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the N-terminal domain was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess ethanol inhibition of wild-type and mutant receptors lacking the N-terminal domain.
Results
As compared to wild-type GluN1/GluN2A receptors, ethanol inhibition was slightly greater in cells expressing GluN2A subunits lacking the N-terminal domain. In contrast, GluN2B N-terminal deletion mutants showed normal ethanol inhibition while those lacking the N-terminal domain in both GluN1 and GluN2B subunits had decreased ethanol inhibition as compared to wild-type receptors. N-terminal domain lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to ethanol.
Conclusions
These findings indicate that the N-terminal domain modestly influences the ethanol sensitivity of NMDA receptors in a subunit-dependent manner. They also show that ifenprodil’s actions on GluN2B containing receptors can be dissociated from those of ethanol. These results suggest that while the N-terminal domain is not a primary site of action for ethanol on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties.
doi:10.1111/acer.12168
PMCID: PMC3812356  PMID: 23905549
Ethanol; NMDA receptors; N-terminal domain; glutamate
6.  Correlated changes in NMDA receptor phosphorylation, functional activity, and sedation by chronic ethanol consumption 
Journal of neurochemistry  2010;115(5):1112-1122.
Alcohol abuse leads to tolerance, dependence and memory impairments that involve excitatory glutamatergic NMDA synaptic transmission. The NMDA receptor is known to undergo activity-dependent adaptive functional changes. Since we observed that acute ethanol inhibition of the NMDA receptor was regulated by protein tyrosine phosphorylation, we investigated the role of protein tyrosine kinases and phosphatases on the NMDA receptor functions by chronic ethanol treatment. We carried out whole-cell recording, Western blotting and behavioral righting reflex measurements to assess the impact of chronic ethanol treatment on NMDA receptor function. Our results indicated that these receptors became resistant to the acute ethanol inhibition following chronic ethanol consumption. This resistance occurred without an increase in the NMDA receptor subunit expression but was associated with decreases in the levels of phospho-Y-1472 NR2B, increases in the levels of STEP33, increases in phospho-p38 mitogen-activated protein kinase (pp38MAPK), and acquisition of tolerance to the sedative effects of ethanol. These data suggested that altered protein tyrosine phosphorylation of the NMDA receptor subunits significantly contributes to functional changes of this receptor by chronic ethanol ingestion. Therefore, preservation of the integrity of tyrosine phosphorylation mechanisms of the NMDA receptor may be important in controlling the progression of alcohol tolerance and dependence.
doi:10.1111/j.1471-4159.2010.06991.x
PMCID: PMC2973329  PMID: 20831600
NR2B; Fyn kinase; protein tyrosine phosphatase; chronic ethanol; hippocampus
7.  Acute Ethanol Exposure Prevents PMA-mediated Augmentation of N-methyl-d-aspartate Receptor Function in Primary Cultured Cerebellar Granule Cells 
Alcohol (Fayetteville, N.Y.)  2011;45(6):595-605.
Many intracellular proteins and signaling cascades contribute to the ethanol sensitivity of native N-methyl-d-aspartate receptors (NMDARs). One putative protein is the serine / threonine kinase, Protein kinase C (PKC). The purpose of this study was to assess if PKC modulates the ethanol sensitivity of native NMDARs expressed in primary cultured cerebellar granule cells (CGCs). With the whole-cell patch-clamp technique, we assessed if ethanol inhibition of NMDA-induced currents (INMDA) (100 μM NMDA plus 10 μM glycine) were altered in CGCs in which the novel and classical PKC isoforms were activated by phorbol-12-myristate-13-acetate (PMA). Percent inhibition by 10, 50 or 100 mM ethanol of NMDA-induced steady-state (ISS) or peak current amplitudes (IPk) of NMDARs expressed in CGCs in which PKC was activated by a 12.5 min, 100 nM PMA exposure at 37° C did not differ from currents obtained from receptors contained in control cells. However, PMA-mediated augmentation of IPk in the absence of ethanol was abolished after brief applications of 10 or 1 mM ethanol co-applied with agonists, and this suppression of enhanced receptor function was observed for up to eight minutes post-ethanol exposure. Because we had previously shown that PMA-mediated augmentation of INMDA of NMDARs expressed in these cells is by activation of PKCα, we assessed the effect of ethanol (1, 10, 50 and 100 mM) on PKCα activity. Ethanol decreased PKCα activity by 18% for 1 mM ethanol and activity decreased with increasing ethanol concentrations with a 50% inhibition observed with 100 mM ethanol. The data suggest that ethanol disruption of PMA-mediated augmentation of INMDA may be due to a decrease in PKCα activity by ethanol. However, given the incomplete blockade of PKCα activity and the low concentration of ethanol at which this phenomenon is observed, other ethanol-sensitive signaling cascades must also be involved.
doi:10.1016/j.alcohol.2011.03.002
PMCID: PMC3154614  PMID: 21624785
N-methyl-d-aspartate receptor; Cerebellar granule cells; Protein Kinase C; Whole-cell patch-clamp; Phorbol 12-myristate 13-acetate; Ethanol
8.  Selective Inhibition by Ethanol of Mitochondrial Calcium Influx Mediated by Uncoupling Protein-2 in Relation to N-Methyl-D-Aspartate Cytotoxicity in Cultured Neurons 
PLoS ONE  2013;8(7):e69718.
Background
We have shown the involvement of mitochondrial uncoupling protein-2 (UCP2) in the cytotoxicity by N-methyl-D-aspartate receptor (NMDAR) through a mechanism relevant to the increased mitochondrial Ca2+ levels in HEK293 cells with acquired NMDAR channels. Here, we evaluated pharmacological profiles of ethanol on the NMDA-induced increase in mitochondrial Ca2+ levels in cultured murine neocortical neurons.
Methodology/Principal Findings
In neurons exposed to glutamate or NMDA, a significant increase was seen in mitochondrial Ca2+ levels determined by Rhod-2 at concentrations of 0.1 to 100 µM. Further addition of 250 mM ethanol significantly inhibited the increase by glutamate and NMDA in Rhod-2 fluorescence, while similarly potent inhibition of the NMDA-induced increase was seen after exposure to ethanol at 50 to 250 mM in cultured neurons. Lentiviral overexpression of UCP2 significantly accelerated the increase by NMDA in Rhod-2 fluorescence in neurons, without affecting Fluo-3 fluorescence for intracellular Ca2+ levels. In neurons overexpressing UCP2, exposure to ethanol resulted in significantly more effective inhibition of the NMDA-induced increase in mitochondrial free Ca2+ levels than in those without UCP2 overexpression, despite a similarly efficient increase in intracellular Ca2+ levels irrespective of UCP2 overexpression. Overexpression of UCP2 significantly increased the number of dead cells in a manner prevented by ethanol in neurons exposed to glutamate. In HEK293 cells with NMDAR containing GluN2B subunit, more efficient inhibition was similarly induced by ethanol at 50 and 250 mM on the NMDA-induced increase in mitochondrial Ca2+ levels than in those with GluN2A subunit. Decreased protein levels of GluN2B, but not GluN2A, subunit were seen in immunoprecipitates with UCP2 from neurons with brief exposure to ethanol at concentrations over 50 mM.
Conclusions/Significance
Ethanol could inhibit the interaction between UCP2 and NMDAR channels to prevent the mitochondrial Ca2+ incorporation and cell death after NMDAR activation in neurons.
doi:10.1371/journal.pone.0069718
PMCID: PMC3713054  PMID: 23874988
9.  The Effects of NR2 Subunit-Dependent NMDA Receptor Kinetics on Synaptic Transmission and CaMKII Activation 
PLoS Computational Biology  2008;4(10):e1000208.
N-Methyl-d-aspartic acid (NMDA) receptors are widely expressed in the brain and are critical for many forms of synaptic plasticity. Subtypes of the NMDA receptor NR2 subunit are differentially expressed during development; in the forebrain, the NR2B receptor is dominant early in development, and later both NR2A and NR2B are expressed. In heterologous expression systems, NR2A-containing receptors open more reliably and show much faster opening and closing kinetics than do NR2B-containing receptors. However, conflicting data, showing similar open probabilities, exist for receptors expressed in neurons. Similarly, studies of synaptic plasticity have produced divergent results, with some showing that only NR2A-containing receptors can drive long-term potentiation and others showing that either subtype is capable of driving potentiation. In order to address these conflicting results as well as open questions about the number and location of functional receptors in the synapse, we constructed a Monte Carlo model of glutamate release, diffusion, and binding to NMDA receptors and of receptor opening and closing as well as a model of the activation of calcium-calmodulin kinase II, an enzyme critical for induction of synaptic plasticity, by NMDA receptor-mediated calcium influx. Our results suggest that the conflicting data concerning receptor open probabilities can be resolved, with NR2A- and NR2B-containing receptors having very different opening probabilities. They also support the conclusion that receptors containing either subtype can drive long-term potentiation. We also are able to estimate the number of functional receptors at a synapse from experimental data. Finally, in our models, the opening of NR2B-containing receptors is highly dependent on the location of the receptor relative to the site of glutamate release whereas the opening of NR2A-containing receptors is not. These results help to clarify the previous findings and suggest future experiments to address open questions concerning NMDA receptor function.
Author Summary
Information processing in the brain is carried out by networks of neurons connected by synapses. Synapses can change strength, allowing these networks to adapt and learn, in a process known as synaptic plasticity. At a synapse, an electrical signal in one neuron is converted into a chemical signal, carried by a neurotransmitter, which is in turn converted into electrical and chemical signals in another neuron by specialized proteins called receptors. One such protein, the N-methyl-d-aspartic acid (NMDA) receptor, is particularly important for plasticity, due to its ability to detect the voltage of the cell receiving the neurotransmitter signal and to the fact that it allows calcium, an important signaling molecule, to enter the cell. Here we use computational modeling to investigate the role of one part of the NMDA receptor: the NR2 subunit. The subunit has various forms, and which of these forms are present in the NMDA receptor can strongly affect the kinetics and other properties of the receptor. We show that, along with changing the kinetics of the receptor, changing the NR2 subunit affects the reliability of the receptor, its ability to respond to large stimuli, and its spatial response properties. These results have implications for synaptic transmission and plasticity.
doi:10.1371/journal.pcbi.1000208
PMCID: PMC2563690  PMID: 18974824
10.  Coactivation of NMDA receptors by glutamate and -serine induces dilation of isolated middle cerebral arteries 
N-methyl--aspartate (NMDA) receptors are glutamate-gated cation channels that mediate excitatory neurotransmission in the central nervous system. In addition to glutamate, NMDA receptors are also activated by coagonist binding of the gliotransmitter, -serine. Neuronal NMDA receptors mediate activity-dependent blood flow regulation in the brain. Our objective was to determine whether NMDA receptors expressed by brain endothelial cells can induce vasodilation of isolated brain arteries. Adult mouse middle cerebral arteries (MCAs) were isolated, pressurized, and preconstricted with norepinephrine. N-methyl--aspartate receptor agonists, glutamate and NMDA, significantly dilated MCAs in a concentration-dependent manner in the presence of -serine but not alone. Dilation was significantly inhibited by NMDA receptor antagonists, -2-amino-5-phosphonopentanoate and 5,7-dichlorokynurenic acid, indicating a response dependent on NMDA receptor glutamate and -serine binding sites, respectively. Vasodilation was inhibited by denuding the endothelium and by selective inhibition or genetic knockout of endothelial nitric oxide synthase (eNOS). We also found evidence for expression of the pan-NMDA receptor subunit, NR1, in mouse primary brain endothelial cells, and for the NMDA receptor subunit NR2C in cortical arteries in situ. Overall, we conclude that NMDA receptor coactivation by glutamate and -serine increases lumen diameter in pressurized MCA in an endothelial and eNOS-dependent mechanism.
doi:10.1038/jcbfm.2011.161
PMCID: PMC3293118  PMID: 22068228
-serine; eNOS; glutamate; middle cerebral artery; NMDA receptor; NR2C
11.  Identification of a single amino acid in GluN1 that is critical for glycine-primed internalization of NMDA receptors 
Molecular Brain  2013;6:36.
Background
NMDA receptors are ligand-gated ion channels with essential roles in glutamatergic synaptic transmission and plasticity in the CNS. As co-receptors for glutamate and glycine, gating of the NMDA receptor/channel pore requires agonist binding to the glycine sites, as well as to the glutamate sites, on the ligand-binding domains of the receptor. In addition to channel gating, glycine has been found to prime NMDA receptors for internalization upon subsequent stimulation of glutamate and glycine sites.
Results
Here we address the key issue of identifying molecular determinants in the glycine-binding subunit, GluN1, that are essential for priming of NMDA receptors. We found that glycine treatment of wild-type NMDA receptors led to recruitment of the adaptor protein 2 (AP-2), and subsequent internalization after activating the receptors by NMDA plus glycine. However, with a glycine-binding mutant of GluN1 – N710R/Y711R/E712A/A714L – we found that treating with glycine did not promote recruitment of AP-2 nor were glycine-treated receptors internalized when subsequently activated with NMDA plus glycine. Likewise, GluN1 carrying a single point mutation – A714L – did not prime upon glycine treatment. Importantly, both of the mutant receptors were functional, as stimulating with NMDA plus glycine evoked inward currents.
Conclusions
Thus, we have identified a single amino acid in GluN1 that is critical for priming of NMDA receptors by glycine. Moreover, we have demonstrated the principle that while NMDA receptor gating and priming share a common requirement for glycine binding, the molecular constraints in GluN1 for gating are distinct from those for priming.
doi:10.1186/1756-6606-6-36
PMCID: PMC3846451  PMID: 23941530
NMDA Receptors; Glycine; Internalization; Endocytosis; Dynamin; GluN1; GluN2
12.  Scaffolding of Fyn Kinase to the NMDA Receptor Determines Brain Region Sensitivity to Ethanol 
Alcohol (ethanol) abuse is a major societal problem. Although ethanol is a structurally simple, diffusible molecule, its sites of action are surprisingly selective, and the molecular mechanisms underlying specificity in ethanol actions are not understood. The NMDA receptor channel is one of the main targets for ethanol in the brain. We report here that the brain region-specific compartmentalization of Fyn kinase determines NMDA receptor sensitivity to ethanol. We demonstrate that, in the hippocampus but not in the cerebral cortex, Fyn is targeted to the NR2B subunit of the NMDA receptor by the scaffolding protein RACK1. During acute exposure to ethanol, RACK1 is dissociated from the complex, thereby facilitating Fyn-mediated phosphorylation of NR2B, which enhances channel activity, counteracting the inhibitory actions of ethanol. In this way, the selective scaffolding can account for the ethanol-induced acute tolerance of NMDA receptor activity that is detected in the hippocampus but not in the cerebral cortex. The phosphorylation-dependent, region-specific activities of ethanol on the NMDA receptor provide a compelling molecular explanation that accounts for the selective activities of ethanol and may have important implications for elucidating pathways leading to alcohol addiction.
PMCID: PMC1262669  PMID: 12736333
ethanol; NMDA; RACK1; Fyn; tyrosine phosphorylation; scaffolding proteins
13.  TNFα-induced neutral sphingomyelinase-2 modulates synaptic plasticity by controlling the membrane insertion of NMDA receptors 
Journal of neurochemistry  2009;109(5):1237-1249.
The insertion and removal of N-methyl D-aspartate (NMDA) receptors from the synapse are critical events that modulate synaptic plasticity. While a great deal of progress has been made on understanding the mechanisms that modulate trafficking of NMDA receptors, we do not currently understand the molecular events required for the fusion of receptor containing vesicles with the plasma membrane. Here we show that sphingomyelin phosphodiesterase3 (also known as neutral sphingomyelinase-2; nSMase2) is critical for TNFα-induced trafficking of NMDA receptors and synaptic plasticity. TNFα initiated a rapid increase in ceramide that was associated with increased surface localization of NMDA receptor NR1 subunits and a specific clustering of NR1 phosphorylated on serines 896 and 897 into lipid rafts. Brief applications of TNFα increased the rate and amplitude of NMDA-evoked calcium bursts and enhanced excitatory postsynaptic currents (EPSCs). Pharmacological inhibition or genetic mutation of nSMase2 prevented TNFα-induced generation of ceramide, phosphorylation of NR1 subuints, clustering of NR1, enhancement of NMDA-evoked calcium flux and EPSCs.
doi:10.1111/j.1471-4159.2009.06038.x
PMCID: PMC2688711  PMID: 19476542
14.  Alcohol Related Changes in Regulation of NMDA Receptor Functions 
Current Neuropharmacology  2008;6(1):39-54.
Long-term alcohol exposure may lead to development of alcohol dependence in consequence of altered neurotransmitter functions. Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action. Several studies showed that ethanol potently inhibits NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “up-regulation” of NMDAR mediated functions. Therefore, alterations in NMDAR function are supposed to contribute to the development of ethanol tolerance, dependence as well as to the acute and late signs of ethanol withdrawal.
A number of publications report alterations in the expression and phosphorylation states of NMDAR subunits, in their interaction with scaffolding proteins or other receptors in consequence of chronic ethanol treatment. Our knowledge on the regulatory processes, which modulate NMDAR functions including factors altering transcription, protein expression and post-translational modifications of NMDAR subunits, as well as those influencing their interactions with different regulatory proteins or other downstream signaling elements are incessantly increasing. The aim of this review is to summarize the complex chain of events supposedly playing a role in the up-regulation of NMDAR functions in consequence of chronic ethanol exposure.
doi:10.2174/157015908783769662
PMCID: PMC2645546  PMID: 19305787
Alcohol dependence; NMDA receptor; subunit expression; post-translation modifications; phosphorylation/ dephosphorylation; compartmentalization.
15.  DAPK1 Interaction with NMDA Receptor NR2B Subunits Mediates Brain Damage in Stroke 
Cell  2010;140(2):222.
SUMMARY
N-methyl-D-aspartate (NMDA) receptors constitute a major subtype of glutamate receptors at extra-synaptic sites that link multiple intracellular catabolic processes responsible for irreversible neuronal death. Here, we report that cerebral ischemia recruits death-associated protein kinase 1 (DAPK1) into the NMDA receptor NR2B protein complex in the cortex of adult mice. DAPK1 directly binds with the NMDA receptor NR2B C-terminal tail consisting of amino acid 1292–1304 (NR2BCT). A constitutively active DAPK1 phosphorylates NR2B subunit at Ser-1303 and in turn enhances the NR1/NR2B receptor channel conductance. Genetic deletion of DAPK1 or administration of NR2BCT that uncouples an activated DAPK1 from an NMDA receptor NR2B subunit in vivo in mice blocks injurious Ca2+ influx through NMDA receptor channels at extrasynaptic sites and protects neurons against cerebral ischemic insults. Thus, DAPK1 physically and functionally interacts with the NMDA receptor NR2B subunit at extra-synaptic sites and this interaction acts as a central mediator for stroke damage.
doi:10.1016/j.cell.2009.12.055
PMCID: PMC2820131  PMID: 20141836
16.  Chronic Ethanol Ingestion Modulates Pro-anxiety Factors Expressed in Rat Central Amygdala 
Alcohol (Fayetteville, N.Y.)  2005;36(2):83-90.
Withdrawal anxiety following chronic ethanol is often associated with relapse in recovering alcoholics. It is likely that brain regions regulating anxiety-like behaviors adapt during chronic ethanol to ultimately regulate such behaviors. The central amygdala contains numerous neurotransmitter systems that have been implicated in the regulation of anxiety-like behavior, including corticotropin releasing factor (CRF) and N-methyl-D-Aspartate (NMDA)-type glutamate receptors. Chronic ethanol exposure causes functional adaptations in both CRF and NMDA receptors that are likely to regulate anxiety-like behaviors expressed during withdrawal. However, the molecular mechanisms governing these adaptations remain un-explored. We therefore evaluated these neurotransmitter systems in Sprague-Dawley rats during chronic ingestion of an ethanol-containing liquid diet. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that pre-proCRF mRNA was significantly up-regulated by chronic ethanol exposure while CRF binding protein mRNA expression did not change. There were also no significant changes observed in any of the NMDA subunit mRNAs, although there was a trend toward greater NR2A mRNA expression during chronic ethanol. Using Western blotting analysis we measured NMDA receptor subunit protein expression. Chronic ethanol exposure did not affect protein levels of the NR1 and NR2B subunits. Like the mRNA measures, chronic ethanol did influence NR2A protein levels but the effects were modest. Our results demonstrate that NMDA receptor subunit mRNA and protein expression are not strongly influenced by exposure to chronic ethanol. This suggests that the functional NMDA receptor adaptations identified by previous studies (Roberto et al., 2004) are likely to be mediated by post-translational events. In contrast, enhanced levels of CRF during/after chronic ethanol are likely to be mediated by increased levels of pre-proCRF mRNA. Together, our findings suggest that adaptations to chronic ethanol by pro-anxiety factors expressed in the central nucleus appear to be mediated by distinct cellular and molecular mechanisms.
doi:10.1016/j.alcohol.2005.07.004
PMCID: PMC1557647  PMID: 16396741
corticotropin releasing factor; NMDA; central amygdala; real-time RT-PCR; western analysis; chronic ethanol
17.  Chronic ethanol up-regulates the synaptic expression of the nuclear translational regulatory protein AIDA-1 in primary hippocampal neurons 
Alcohol (Fayetteville, N.Y.)  2012;46(6):569-576.
Recent studies have identified synaptic proteins that undergo synapse-to-nucleus translocation in response to neuronal activity that modulate protein synthesis. One such translational regulatory protein of the postsynaptic density (PSD) is AIDA-1d, which binds to PSD-95 via its C-terminus. Activation of synaptic NMDA receptors induces the cleavage of AIDA-1d, and the N-terminus is then shuttled to nuclear Cajal bodies where it plays a role in regulating global protein synthesis. Chronic ethanol exposure has been shown to increase the synaptic clustering of NMDA receptors and PSD-95. Here, we tested the hypotheses that AIDA-1d regulates chronic ethanol-induced increases in synaptic NMDA receptor expression. As reported, we found that AIDA-1 was highly enriched in dendritic spines and co-localized with PSD-95. Acute NMDA treatment increased AIDA-1 colocalization with p80 coilin, a marker of Cajal bodies. Chronic treatment (4 day) of cultures with ethanol (25 – 100 mM) or with the NMDA receptor antagonist AP-V (50 µM) enhanced the clustering of AIDA-1 at synaptic sites. However, chronic ethanol treatment (50 mM) in the presence of the NMDA receptor agonist NMDA (2.5 µM) prevented this increase. Surprisingly, PSD-95 did not seem to play a role in the synaptic distribution of AIDA-1 as this distribution was not affected by declustering PSD-95 from synapses in response to inhibition of palmitoylation. We found that lentiviral knockdown of AIDA-1d did not affect protein expression levels of NMDA receptor subunits GluN1, GluN1 C2’, or GluN2B. The results of this study demonstrate that synaptic AIDA-1 expression is enhanced by chronic ethanol exposure that can be prevented by concurrent stimulation of NMDA receptors. In addition, we found that the association of AIDA-1 with PSD-95 is not required for its localization to the PSD. Moreover, we found that AIDA-1 does not regulate protein expression levels or alternative splicing of the GluN1 subunit of NMDA receptors.
doi:10.1016/j.alcohol.2012.04.004
PMCID: PMC3411901  PMID: 22703994
AIDA-1, chronic ethanol, synapse-to-nucleus signaling; NMDA receptors; Cajal bodies; PSD-95
18.  Activation of NMDA receptors leads to phosphorylation of TRPV1 S800 by protein kinase C and A-Kinase anchoring protein 150 in rat trigeminal ganglia 
A-Kinase anchoring protein 150 (AKAP150) is required for the phosphorylation of transient receptor potential cation channel subfamily V member 1 (TRPV1) by PKA or PKC in sensory neurons and, hence, affects TRPV1-dependent hyperalgesia under pathological conditions. Recently, we showed that the activation of N-methyl-d-aspartate (NMDA) receptors sensitizes TRPV1 by enhancing serine phosphorylation through PKC in trigeminal nociceptors. In this study, we extended this observation by investigating whether AKAP150 mediates NMDA-induced phosphorylation of TRPV1 via PKC in native sensory neurons in the rat. By adopting a phospho-specific antibody combined with a surface biotinylation assay, we first assessed NMDA-induced changes in the phosphorylation level of serine 800 residues (S800) in TRPV1 delimited to cell surface membrane in cultured trigeminal ganglia (TG). The biotinylation assay yielded that the application of NMDA significantly increased the phosphorylation of S800 (p-S800) of TRPV1 at time points correlating with the development of NMDA-induced mechanical hyperalgesia [10]. We then obtained a siRNA sequence against AKAP150 that dose-dependently down-regulated the AKAP150 protein. Pretreatment of TG culture with the siRNA, but not mismatch sequences, prevented the NMDA-induced phosphorylation of serine residues of total TRPV1 as well as S800 of membrane bound TRPV1. We confirmed that AKAP150 coimmunoprecipitated with TRPV1 and demonstrated that it also co-immunoprecipitated with NMDA receptor subunits (NR1 and NR2B) in TG. These data offer novel information that the activation of NMDA-induced TRPV1 sensitization involves p-S800 of TRPV1 in cell surface membrane in native sensory neurons and that AKAP150 is required for NMDA-and PKC-mediated phosphorylation of TRPV1 S800. Therefore, we propose that the NMDA receptor, AKAP150, and TRPV1 forms a signaling complex that underlies the sensitization of trigeminal nociceptors by modulating phosphorylation of specific TRPV1 residues.
doi:10.1016/j.bbrc.2012.07.008
PMCID: PMC3408820  PMID: 22789851
Nociceptor; Sensitization; Peripheral; Rat
19.  Role of Altered Structure and Function of NMDA Receptors in Development of Alcohol Dependence 
Current Neuropharmacology  2005;3(4):281-297.
Long-term alcohol exposure gives rise to development of physical dependence on alcohol in consequence of changes in certain neurotransmitter functions. Accumulating evidence suggests that the glutamatergic neurotransmitter system, especially the N-methyl-D-aspartate (NMDA) type of glutamate receptors is a particularly important site of ethanol’s action, since ethanol is a potent inhibitor of the NMDA receptors (NMDARs) and prolonged ethanol exposition leads to a compensatory “upregulation” of NMDAR mediated functions supposedly contributing to the occurrence of ethanol tolerance, dependence as well as the acute and delayed signs of ethanol withdrawal.
Recently, expression of different types of NMDAR subunits was found altered after long-term ethanol exposure. Especially, the expression of the NR2B and certain splice variant forms of the NR1 subunits were increased in primary neuronal cultures treated intermittently with ethanol. Since NMDA ion channels with such an altered subunit composition have increased permeability for calcium ions, increased agonist sensitivity, and relatively slow closing kinetics, the abovementioned alterations may underlie the enhanced NMDAR activation observed after long-term ethanol exposure. In accordance with these changes, the inhibitory potential of NR2B subunit-selective NMDAR antagonists is also increased, demonstrating excellent potency against alcohol withdrawal-induced in vitro cytotoxicity. Although in vivo data are few with these compounds, according to the effectiveness of the classic NMDAR antagonists in attenuation, not only the physical symptoms, but also some affective and motivational components of alcohol withdrawal, novel NR2B subunit selective NMDAR antagonists may offer a preferable alternative in the pharmacotherapy of alcohol dependence.
PMCID: PMC2268999  PMID: 18369402
Alcohol; dependence; withdrawal; NMDA receptor; NR2B subunit selective antagonist; pharmacotherapy
20.  Fyn Kinase and NR2B-Containing NMDA Receptors Regulate Acute Ethanol Sensitivity But Not Ethanol Intake or Conditioned Reward 
Background:
The tyrosine kinase Fyn previously has been shown to play a key role in mediating acute tolerance to ethanol. Recently, we found that the compartmentalization of Fyn to the NR2B subunit of the NMDA receptor (NMDAR) in the hippocampus regulates Fyn phosphorylation of NR2B in response to ethanol, which mediates the acute tolerance of NMDAR to ethanol inhibition in hippocampal slices. In this study we determined, first, whether acute tolerance to ethanol inhibition is mediated via NR2B-containing NMDARs in vivo and, second, whether the increase in acute sensitivity to ethanol in the Fyn−/− mice influences ethanol consumption or ethanol’s conditioned rewarding effects.
Methods:
A loss of righting reflex test was used to study the acute/sedative effects of ethanol after intraperitoneal injections of sedative doses of ethanol. Conditioned place preference was used to study the rewarding properties of ethanol. The two-bottle choice protocol was used to measure oral ethanol self-administration and preference as described previously.
Results:
We found that systemic injection of the NR2B-containing NMDAR selective antagonist, ifenprodil, abolished the differences between Fyn+/+ and Fyn−/− mice in sensitivity to the acute sedative effects of ethanol. Moreover, we found that Fyn−/− and Fyn+/+ mice did not differ in their voluntary ethanol consumption or in the rewarding properties of ethanol.
Conclusions:
Our results suggest that the interaction between Fyn and NR2B mediates the acute sedative effects of ethanol, and that alteration in acute ethanol sensitivity does not necessarily correlate with levels of ethanol consumption or the rewarding properties of ethanol.
doi:10.1097/01.ALC.0000095924.87729.D8
PMCID: PMC1193705  PMID: 14634488
Fyn; NR2B; Phosphorylation; Ethanol; Acute Sensitivity; Self-Administration
21.  Anesthetic-Like Modulation of Receptor Function by Surfactants: A Test of the Interfacial Theory of Anesthesia 
Anesthesia and analgesia  2008;107(3):868-874.
Introduction
Inhaled anesthetics are interfacially active, concentrating at interfaces such as the protein/water or bilayer/water interfaces. We tested the hypothesis that interfacial activity was a sufficient condition for anesthetic-like modulation of receptor function by applying surfactants to γ-aminobutyric acid type A (GABAA), glycine, and N-methyl-D-aspartate (NMDA) receptors. We defined anesthetic-like modulation as an increase in currents through native channels that isoflurane and ethanol increased currents through, and a decrease in currents through channels that isoflurane and ethanol decreased currents through. We also tested the null hypothesis that there would be no difference in modulation of channel currents by surfactants in receptors with point mutations that diminished their response to isoflurane and ethanol compared to the native version of these receptors.
Methods
The effect of seven surfactants with different head group charges (anionic, cationic, zwitterionic, and uncharged) and tail lengths (eight carbons and twelve carbons) on homomeric wild type α1 and mutant α1 (S267I) glycine receptors, wild type α1β2γ2s and mutant α1(S270I)β2γ2s GABAA receptors, and wild type NR1/NR2A and mutant NR1(F639A)/NR2A NMDA receptors was studied. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping.
Results
All seven surfactants, isoflurane, and ethanol potentiated GABAA receptor function. Six of seven surfactants, isoflurane, and ethanol potentiated glycine receptor function. Six of seven surfactants, isoflurane, and ethanol inhibited NMDA receptor function. For the mutant receptors, five of seven surfactants increased currents through GABAA receptors, while six of seven surfactants increased currents through glycine receptors. Six of seven surfactants decreased currents through the NMDA receptor. In contrast to isoflurane and ethanol, surfactants as a group did not diminish modulation of mutant compared to wild type receptors.
Conclusion
These findings identify another large class of compounds (surfactants) that modulate the function of GABAA, glycine, and NMDA receptors in a manner that is qualitatively similar to inhaled anesthetics. We cannot reject the hypothesis that interfacial activity is a sufficient condition for anesthetic-like modulation of these receptors. Mutations that diminish the modulatory effect of isoflurane and ethanol did not diminish the modulatory effect of the surfactants.
doi:10.1213/ane.0b013e31817ee500
PMCID: PMC2668573  PMID: 18713898
Anesthesia; GABAA receptor; Glycine receptor; NMDA receptor; detergent; surface-active agents; interfacial activity
22.  Two serine residues on GluN2A C-terminal tails control NMDA receptor current decay times 
Channels  2013;7(2):126-132.
NMDA receptors are glutamate-activated, Ca2+-permeable ion channels with critical roles in synaptic transmission and plasticity. The shape and size of their current is modulated by several kinase/phosphatase systems, and numerous residues located on the receptors’ intracellular C-termini are phosphorylated in vivo. To investigate the mechanisms by which phosphorylation may control channel gating, we examined the single-channel behaviors of receptors carrying the S900A or S929A substitution in their GluN2A subunits and thus were rendered resistant to phosphorylation at those sites. We found that the mutations reduced channel open probability primarily by increasing the frequency of desensitized events. The kinetic models we developed revealed complex but similar changes in mechanism for the two mutants, leading to the view that dephosphorylation at either site may cause receptors to activate slower, deactivate faster and desensitize more frequently. This modulatory mechanism is consistent with the proposed roles for these residues in Ca2+-dependent desensitization and calcineurin-mediated reduction of current during brain development.
doi:10.4161/chan.23968
PMCID: PMC3667882  PMID: 23503100
ionotropic glutamate receptors; NMDA receptors; reaction mechanism; single-channel kinetics; protein phosphorylation
23.  NMDA receptors in primary afferents require phosphorylation by Src family kinases to induce substance P release in the rat spinal cord 
Neuroscience  2010;166(3):924-934.
The function of NMDA receptors in primary afferents remains controversial, in particular regarding their ability to evoke substance P release in the spinal cord. The objective of this study was, first, to confirm that substance P release evoked by NMDA is mediated by NMDA receptors in primary afferent terminals. Second, we investigated whether these NMDA receptors are inactivated in some conditions, which would explain why their effect on substance P release was not observed in some studies. Substance P release was induced in spinal cord slices and measured as NK1 receptor internalization in lamina I neurons. NMDA (combined with D-serine) induced NK1 receptor internalization with an EC50 of 258 nM. NMDA-induced NK1 receptor internalization was abolished by the NK1 receptor antagonist L-703,606, confirming that is was caused by substance P release, by NMDA receptor antagonists (MK1801 and ifenprodil), showing that it was mediated by NMDA receptors containing the NR2B subunit, and by preincubating the slices with capsaicin, showing that the substance P release was from primary afferents. However, it was not affected by lidocaine and ω-conotoxin MVIIA, which block Na+ channels and voltage-dependent Ca2+ channels, respectively. Therefore, NMDA-induced substance P release does not require firing of primary afferents or the opening of Ca2+ channels, which is consistent with the idea that NMDA receptors induce substance P directly by letting Ca2+ into primary afferent terminals. Importantly, NMDA-induced substance P release was eliminated by preincubating the slices for one hour with the Src family kinase inhibitors PP1 and dasatinib, and was substantially increased by the protein tyrosine phosphatase inhibitor BVT948. In contrast, PP1 did not affect NK1 receptor internalization induced by capsaicin. These results show that tyrosine-phosphorylation of these NMDA receptors is regulated by the opposite actions of Src family kinases and protein tyrosine phosphatases, and is required to induce substance P release.
doi:10.1016/j.neuroscience.2010.01.009
PMCID: PMC2837134  PMID: 20074620
C-fiber; dorsal horn; internalization; nociceptor; neurokinin-1 receptor; protein tyrosine phosphatase
24.  Ethanol disrupts NMDA receptor and astroglial EAAT2 modulation of Kv2.1 potassium channels in hippocampus 
Alcohol (Fayetteville, N.Y.)  2009;43(1):45-50.
Delayed-rectifier Kv2.1 channels are the principal component of voltage-sensitive K+ currents (IK) in hippocampal neurons and are critical regulators of somatodendritic excitability. In a recent study, we demonstrated that surface trafficking and phosphorylation of Kv2.1 channels is modulated by NMDA-type glutamate receptors and that astroglial excitatory amino acid transporters 2 (EAAT2) regulate the coupling of NMDA receptors and Kv2.1 channels. Since ethanol is known to acutely inhibit NMDA receptors, we sought to determine if NMDA receptor and astroglial EAAT2 modulation of Kv2.1 channels is impaired by ethanol in rodent hippocampus. As expected, bath application of NMDA to hippocampal cultures reduced the size of Kv2.1 clusters and produced a hyperpolarizing shift in the voltage-dependent activation of IK that was associated with dephosphorylated Kv2.1 channels. Ethanol, applied acutely, prevented the hyperpolarizing shift in activation of IK induced by NMDA and restored Kv2.1 clustering and phosphorylation to near control levels. Ethanol also attenuated the dephosphorylation of Kv2.1 channels produced by the EAAT2 selective inhibitor dihydrokainic acid. These data demonstrate that acute ethanol disrupts changes in Kv2.1 channels that follow NMDA receptor activation and impairs astroglial regulation of the functional coupling between NMDA receptors and Kv2.1 channels.
doi:10.1016/j.alcohol.2008.10.001
PMCID: PMC2674284  PMID: 19185209
Kv2.1 channels; NMDA receptors; ethanol; astroglial EAAT2; phosphorylation; clustering
25.  Downregulation of Src-kinase and glutamate-receptor phosphorylation after traumatic brain injury 
Phosphorylation of N-methyl-D-aspartate (NMDA) receptors is a major regulatory mechanism underlying synaptic plasticity. However, changes in NMDA receptors and phosphorylation after traumatic brain injury (TBI) remain incompletely understood. Using an animal TBI model, we observed that the protein level of NMDA receptor subunit NR2B was downregulated in synaptosomal fractions obtained from the ipsilateral neocortical injury region, whereas the levels of NR2A, NR1, and PSD93 were not significantly altered at 4 and 24 hours after TBI. Further investigation showed that tyrosine phosphorylations of NR2B Y1472 and PSD93 Y340 in synaptosomal fractions were significantly decreased relative to their total protein level after TBI. Correspondingly, phosphorylation of the Src-kinase-inhibitory site Y527 was increased, whereas phosphorylation of the activation site Y416 was decreased, indicating that the activity of Src kinase is significantly inhibited after TBI. In comparison, other Src family kinase substrates of NMDA receptor, NR2A Y1246, NR2A Y1325, and NR2B Y1070 were not obviously affected after TBI. The results suggest that TBI downregulates the Src-kinase-mediated phosphorylation of NR2 and PSD93 to destabilize the synaptic localization of NMDA receptors. Therefore, post-TBI loss of NMDA receptors may contribute to the depression of synaptic activity after TBI.
doi:10.1038/jcbfm.2013.121
PMCID: PMC3790935  PMID: 23838828
NMDA receptor; PSD93; Src family kinases; traumatic brain injury; tyrosine phosphorylation

Results 1-25 (847908)