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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.  Involvement of protein kinase C and Src tyrosine kinase in acute tolerance to ethanol inhibition of spinal NMDA-induced pressor responses in rats 
British Journal of Pharmacology  2009;158(3):806-818.
Background and purpose:
The present study was carried out to examine the role of protein kinases in the development of acute tolerance to the effects of ethanol on spinal N-methyl-D-aspartate (NMDA) receptor-mediated pressor responses during prolonged ethanol exposure.
Experimental approach:
Blood pressure responses induced by intrathecal injection of NMDA were recorded. The levels of several phosphorylated residues on NMDA receptor NR1 (GluN1) (NR1) and NMDA receptor NR2B (GluN2B) (NR2B) subunits were determined by immunohistochemistry and Western blot analysis.
Key results:
Ethanol inhibited spinal NMDA-induced pressor responses at 10 min, but the inhibition was significantly reduced at 40 min following continuous infusion. This effect was dose-dependently blocked by chelerythrine [a protein kinase C (PKC) inhibitor, 1–1000 pmol] or PP2 (a Src family tyrosine kinase inhibitor, 1–100 pmol) administered intrathecally 10 min following ethanol infusion. A significant increase in the immunoreactivity of phosphoserine 896 of NR1 subunits (pNR1-Ser896) and phosphotyrosine 1336 of NR2B subunits (pNR2B-Tyr1336) was found in neurons of intermediolateral cell column during the development of tolerance. Levels of pNR1-Ser896 and pNR2B-Tyr1336 were also significantly increased in lateral horn regions of the spinal cord slices incubated with ethanol for 40 min in vitro. The increases in pNR1-Ser896 and pNR2B-Tyr1336 levels were inhibited by post-treatment with chelerythrine and PP2, respectively, both in the in vivo and in vitro studies.
Conclusions and implications:
The results suggest that activation of PKC and Src tyrosine kinase during prolonged ethanol exposure leading to increases in the levels of pNR1-Ser896 and pNR2B-Tyr1336 may contribute to acute tolerance to inhibition by ethanol of NMDA receptor function.
doi:10.1111/j.1476-5381.2009.00364.x
PMCID: PMC2765600  PMID: 19703167
alcohol; NMDA receptor; protein kinase C; Src tyrosine kinases; ion channel regulation; tolerance; sympathetic activity
7.  Differential actions of ethanol and trichloroethanol at sites in the M3 and M4 domains of the NMDA receptor GluN2A (NR2A) subunit 
British Journal of Pharmacology  2009;158(5):1395-1404.
Background and purpose:
Alcohol produces its behavioural effects in part due to inhibition of N-methyl-d-aspartate (NMDA) receptors in the CNS. Previous studies have identified amino acid residues in membrane-associated domains 3 (M3) and 4 (M4) of the NMDA receptor that influence ethanol sensitivity. In addition, in other alcohol-sensitive ion channels, sedative-hypnotic agents have in some cases been shown to act at sites distinct from the sites of ethanol action. In this study, we compared the influence of mutations at these sites on sensitivity to ethanol and trichloroethanol, a sedative-hypnotic agent that is a structural analogue of ethanol.
Experimental approach:
We constructed panels of mutants at ethanol-sensitive positions in the GluN2A (NR2A) NMDA receptor subunit and transiently expressed these mutants in human embryonic kidney 293 cells. We used whole-cell patch-clamp recording to assess the actions of ethanol and trichloroethanol in these mutant NMDA receptors.
Key results:
Ethanol sensitivity of mutants at GluN2A(Ala825) was not correlated with any physicochemical measures tested. Trichloroethanol sensitivity was altered in two of three ethanol-insensitive mutant GluN2A subunits: GluN2A(Phe637Trp) in M3 and GluN2A(Ala825Trp) in M4, but not GluN2A(Met823Trp). Trichloroethanol sensitivity decreased with increasing molecular volume at Phe637 or increasing hydrophobicity at Ala825 and was correlated with ethanol sensitivity at both sites.
Conclusions and implications:
Evidence obtained to date is consistent with a role of GluN2A(Ala825) as a modulatory site for ethanol and trichloroethanol sensitivity, but not as a binding site. Trichloroethanol appears to inhibit the NMDA receptor in a manner similar, but not identical to, that of ethanol.
doi:10.1111/j.1476-5381.2009.00397.x
PMCID: PMC2782349  PMID: 19788495
glutamate receptor; alcohol; sedative-hypnotic; membrane-associated domains; electrophysiology; mutant
8.  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
9.  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
10.  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
11.  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
12.  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
13.  Chronic nicotine treatment differentially modifies acute nicotine and alcohol actions on GABAA and glutamate receptors in hippocampal brain slices 
British Journal of Pharmacology  2011;162(6):1351-1363.
BACKGROUND AND PURPOSE
Tobacco and alcohol are often co-abused producing interactive effects in the brain. Although nicotine enhances memory while ethanol impairs it, variable cognitive changes have been reported from concomitant use. This study was designed to determine how nicotine and alcohol interact at synaptic sites to modulate neuronal processes.
EXPERIMENTAL APPROACH
Acute effects of nicotine, ethanol, and both drugs on synaptic excitatory glutamatergic and inhibitory GABAergic transmission were measured using whole-cell recording in hippocampal CA1 pyramidal neurons from brain slices of mice on control or nicotine-containing diets.
KEY RESULTS
Acute nicotine (50 nM) enhanced both GABAergic and glutamatergic synaptic transmission; potentiated GABAA receptor currents via activation of α7* and α4β2* nAChRs, and increased N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor currents through α7* receptors. While ethanol (80 mM) also increased GABAA currents, it inhibited NMDA currents. Although ethanol had no effect on AMPA currents, it blocked nicotine-induced increases in NMDA and AMPA currents. Following chronic nicotine treatment, acute nicotine or ethanol did not affect NMDA currents, while the effects of GABAergic responses were not altered.
CONCLUSIONS AND IMPLICATIONS
Acute ethanol ingestion selectively attenuated nicotine enhancement of excitatory glutamatergic NMDA and AMPA receptor function, suggesting an overall reduction in excitatory output from the hippocampus. It also indicated that ethanol could decrease the beneficial effects of nicotine on memory performance. In addition, chronic nicotine treatment produced tolerance to the effects of nicotine and cross-tolerance to the effects of ethanol on glutamatergic activity, leading to a potential increase in the use of these drugs.
doi:10.1111/j.1476-5381.2010.01141.x
PMCID: PMC3058167  PMID: 21133888
ethanol; alcohol; GABAA receptor; NMDA receptor; AMPA receptor; nicotinic receptor
14.  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
15.  Roles of subunit phosphorylation in regulating glutamate receptor function 
Protein phosphorylation is an important mechanism for regulating ionotropic glutamate receptors (iGluRs). Early studies have established that major iGluR subtypes, including α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and N-methyl-D-aspartate (NMDA) receptors, are subject to phosphorylation. Multiple serine, threonine, and tyrosine residues predominantly within the C-terminal regions of AMPA receptor and NMDA receptor subunits have been identified as sensitive phosphorylation sites. These distinct sites undergo either constitutive phosphorylation or activity-dependent phosphorylation induced by changing cellular and synaptic inputs as reversible events. An increasing number of synapse-enriched protein kinases have been found to phosphorylate iGluR. The common kinases include protein kinase A, protein kinase C, Ca2+/calmodulin-dependent protein kinase II, Src/Fyn non-receptor tyrosine kinases, and cyclin dependent kinase-5. Regulated phosphorylation plays a well-documented role in modulating the biochemical, biophysical, and functional properties of the receptor. In the future, identifying the precise mechanisms how phosphorylation regulates iGluR activities and finding the link between iGluR phosphorylation and the pathogenesis of various brain diseases, including psychiatric and neurodegenerative diseases, chronic pain, stroke, Alzheimer’s disease and substance addiction, will be hot topics and could contribute to the development of novel pharmacotherapies, by targeting the defined phosphorylation process, for suppressing iGluR-related disorders.
doi:10.1016/j.ejphar.2013.11.019
PMCID: PMC3966912  PMID: 24291102
Excitatory amino acid; AMPA; NMDA; PKA; PKC; CaMKII; Cdk5; tyrosine kinase
16.  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
17.  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
18.  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.
19.  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
20.  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
21.  NMDA receptor characterization and subunit expression in rat cultured mesencephalic neurones 
British Journal of Pharmacology  1999;126(1):121-130.
NMDA-induced changes in free intracellular Ca2+ concentration ([Ca2+]i) were determined in individual cultured rat mesencephalic neurones by the fura-2 method. mRNA expression encoding NMDA receptor subunits (NR1, NR2A-D) was examined by RT–PCR.NMDA (1–100 μM, plus 10 μM glycine) induced a concentration-dependent increase in [Ca2+]i (EC50=5.7 μM). The effect of NMDA was virtually insensitive to tetrodotoxin (0.3 μM) and nitrendipine (1 μM), but dependent on extracellular Ca2+. 5,7-Dichlorokynurenic acid (10 μM), a specific antagonist at the glycine binding site on the NMDA receptor, abolished the NMDA response.Memantine, an open-channel blocker, and ifenprodil, a preferential non-competitive NR1/NR2B receptor antagonist diminished the NMDA effect with an IC50 value of 0.17 and 1 μM, respectively. Ethanol at 50 and 100 mM caused about 25 and 45%-inhibition, respectively.Agarose gel analysis of the PCR products followed by ethidium bromide fluorescence or CSPD chemiluminescence detection revealed an almost exclusive expression of the NR1 splice variants lacking exon (E) 5 and E22. The 3′ splice form without both E21 and E22 exceeded that containing E21 by approximately 4 fold. The relative amounts of NR2A, NR2B, NR2C corresponded to approximately 1:2:1. NR2D mRNA was also detectable.In conclusion, mesencephalic neurones bear ethanol-sensitive NMDA receptors which might be involved in the development of ethanol dependence and withdrawal. The high affinity of NMDA to this receptor, its sensitivity to ifenprodil and memantine may suggest that the mesencephalic NMDA receptor comprises the NR1 splice variant lacking E5, NR2B, and NR2C, respectively.
doi:10.1038/sj.bjp.0702284
PMCID: PMC1565789  PMID: 10051128
Cultured mesencephalic neurones; NMDA receptors; intracellular Ca2+; ethanol; ifenprodil; memantine; fura-2 method; RT–PCR; NR1 splice variants; NR2 subunits
22.  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
23.  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
24.  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
25.  State-dependent NMDA receptor antagonism by Ro 8-4304, a novel NR2B selective, non-competitive, voltage-independent antagonist 
British Journal of Pharmacology  1998;123(3):463-472.
Subunit-selective blockade of N-methyl-D-aspartate (NMDA) receptors provides a potentially attractive strategy for neuroprotection in the absence of undesirable side effects. Here, we describe a novel NR2B-selective NMDA antagonist, 4-{3-[4-(4-fluoro-phenyl)-3,6-dihydro-2H-pyridin-1-yl]-2-hydroxy-propoxy}-benzamide (Ro 8-4304), which exhibits >100 fold higher affinity for recombinant NR1001/NR2B than NR1001/NR2A receptors.Ro 8-4304 is a voltage-independent, non-competitive antagonist of NMDA receptors in rat cultured cortical neurones and exhibits a state-dependent mode of action similar to that described for ifenprodil.The apparent affinity of Ro 8-4304 for the NMDA receptor increased in an NMDA concentration-dependent manner so that Ro 8-4304 inhibited 10 and 100 μM NMDA responses with IC50s of 2.3 and 0.36 μM, respectively. Currents elicited by 1 μM NMDA were slightly potentiated in the presence of 10 μM Ro 8-4304, and Ro 8-4304 binding slowed the rate of glutamate dissociation from NMDA receptors.These results were predicted by a reaction scheme in which Ro 8-4304 exhibits a 14 and 23 fold higher affinity for the activated and desensitized states of the NMDA receptor, respectively, relative to the agonist-unbound resting state. Additionally, Ro 8-4304 binding resulted in a 3–4 fold increase in receptor affinity for glutamate site agonists.Surprisingly, whilst exhibiting a similar affinity for NR2B-containing NMDA receptors as ifenprodil, Ro 8-4304 exhibited markedly faster kinetics of binding and unbinding to the NMDA receptor. This spectrum of kinetic behaviour reveals a further important feature of this emerging class of NR2B-selective compounds.
doi:10.1038/sj.bjp.0701634
PMCID: PMC1565193  PMID: 9504387
NMDA receptor antagonists; neuroprotection; glutamate receptors

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