Sensitivity Of N-methyl-D-aspartate Receptors Research Articles

The escalation in ethanol consumption following chronic intermittent ethanol exposure is blunted in mice expressing ethanol-resistant GluN1 or GluN2A NMDA receptor subunits.

N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated ion channels essential for glutamatergic transmission and plasticity. NMDARs are inhibited by acute ethanol and undergo brain region-specific adaptations after chronic alcohol exposure. In previous studies, we reported that knock-in mice expressing ethanol-insensitive GluN1 or GluN2A NMDAR subunits display altered behavioral responses to acute ethanol and genotype-dependent changes in drinking using protocols that do not produce dependence. A key unanswered question is whether the intrinsic ethanol sensitivity of NMDARs also plays a role in determining behavioral adaptations that accompany the development of dependence. To test this, we exposed mice to repeated cycles of chronic intermittent ethanol (CIE) vapor known to produce a robust escalation in ethanol consumption and preference. As expected, wild-type mice showed a significant increase from baseline in ethanol consumption and preference after each of the four weekly CIE cycles. In contrast, ethanol consumption in male GluN2A(A825W) mice was unchanged following cycles 1, 2, and 4 of CIE with a modest increase appearing after cycle 3. Wild-type and GluN2A(A825W) female mice did not show a clear or consistent escalation in ethanol consumption or preference following CIE treatment. In male GluN1(F639A) mice, the increase in ethanol consumption observed with their wild-type littermates was delayed until later cycles of exposure. These results suggest that the acute ethanol sensitivity of NMDARs especially those containing the GluN2A subunit may be a critical factor in the escalation of ethanol intake in alcohol dependence.

Differential effects of TM4 tryptophan mutations on inhibition of N-methyl-d-aspartate receptors by ethanol and toluene.

The voluntary use and abuse of alcohol and inhalants is a recognized health problem throughout the world. Previous studies have shown that these agents affect brain function in a variety of ways including direct inhibition of key ion channels that regulate neuronal excitability. Among these, the N-methyl-d-aspartate (NMDA) receptor is particularly important given its key role in glutamatergic synaptic transmission, neuronal plasticity and learning and memory. Previous studies from this laboratory and others have identified key residues within transmembrane (TM) domains of the NMDA receptor that appear to regulate its sensitivity to alcohol and anesthetics. In this study, we extend these findings and examine the role of a TM4 residue in modulating sensitivity of recombinant NMDA receptors to ethanol and toluene. HEK293 cells were transfected with GluN1-1a and either wild-type or tryptophan-substituted GluN2(A-D) subunits and whole-cell currents were recorded using patch-clamp electrophysiology in the absence or presence of ethanol or toluene. Both ethanol (100mM) and toluene (1 or 3mM) reversibly inhibited glutamate-activated currents from wild-type NMDARs with GluN2B containing receptors showing heightened sensitivity to either agent. Substitution of tryptophan (W) at positions 825, 826, 823 or 850 in the TM4 domain of GluN2A, GluN2B, GluN2C or GluN2D subunits; respectively, significantly reduced the degree of inhibition by ethanol. In contrast, toluene inhibition of glutamate-activated currents in cells expressing the TM4-W mutants was not different from that of the wild-type controls. These data suggest that despite similarities in their action on NMDARs, ethanol and toluene may act at different sites to reduce ion flux through NMDA receptors.

Neurochemistry of Pressure-Induced Nitrogen and Metabolically Inert Gas Narcosis in the Central Nervous System.

Gases that are not metabolized by the organism are thus chemically inactive under normal conditions. Such gases include the "noble gases" of the Periodic Table as well as hydrogen and nitrogen. At increasing pressure, nitrogen induces narcosis at 4 absolute atmospheres (ATAs) and more in humans and at 11 ATA and more in rats. Electrophysiological and neuropharmacological studies suggest that the striatum is a target of nitrogen narcosis. Glutamate and dopamine release from the striatum in rats are decreased by exposure to nitrogen at a pressure of 31 ATA (75% of the anesthetic threshold). Striatal dopamine levels decrease during exposure to compressed argon, an inert gas more narcotic than nitrogen, or to nitrous oxide, an anesthetic gas. Inversely, striatal dopamine levels increase during exposure to compressed helium, an inert gas with a very low narcotic potency. Exposure to nitrogen at high pressure does not change N-methyl-d-aspartate (NMDA) glutamate receptor activities in Substantia Nigra compacta and striatum but enhances gama amino butyric acidA (GABAA) receptor activities in Substantia Nigra compacta. The decrease in striatal dopamine levels in response to hyperbaric nitrogen exposure is suppressed by recurrent exposure to nitrogen narcosis, and dopamine levels increase after four or five exposures. This change, the lack of improvement of motor disturbances, the desensitization of GABAA receptors on dopamine cells during recurrent exposures and the long-lasting decrease of glutamate coupled with the higher sensitivity of NMDA receptors, suggest a nitrogen toxicity induced by repetitive exposures to narcosis. These differential changes in different neurotransmitter receptors would support the binding protein theory. © 2016 American Physiological Society. Compr Physiol 6:1579-1590, 2016.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Mercury-induced neurotoxicity and neuroprotective effects of berberine.

Deletion of the N-Terminal Domain Alters the Ethanol Inhibition ofN-Methyl-d-Aspartate Receptors in a Subunit-Dependent Manner

Ethanol (EtOH) inhibition of N-methyl-d-aspartate (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 (NTD) 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. EtOH 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 NTD may be important in regulating the EtOH sensitivity of NMDA receptors. Wild-type GluN1 and GluN2 subunits and those in which the coding sequence for the NTD was deleted were expressed in HEK293 cells. Whole-cell voltage-clamp recording was used to assess EtOH inhibition of wild-type and mutant receptors lacking the NTD. As compared to wild-type GluN1/GluN2A receptors, EtOH inhibition was slightly greater in cells expressing GluN2A subunits lacking the NTD. In contrast, GluN2B N-terminal deletion mutants showed normal EtOH inhibition while those lacking the NTD in both GluN1 and GluN2B subunits had decreased EtOH inhibition as compared to wild-type receptors. NTD lacking GluN2B receptors were insensitive to ifenprodil but retained normal sensitivity to EtOH. These findings indicate that the NTD modestly influences the EtOH 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 EtOH. These results suggest that while the NTD is not a primary site of action for EtOH on NMDA receptors, it likely affects sensitivity via actions on intrinsic channel properties.

Effects of ethanol on phosphorylation site mutants of recombinant N-methyl- d-aspartate receptors

N-methyl- d-aspartate (NMDA) receptors are ligand-gated ion channels activated by the neurotransmitter glutamate. These channels are highly expressed by brain neurons and are critically involved in excitatory synaptic transmission. Results from previous studies show that both native and recombinant NMDA receptors are inhibited by ethanol at concentrations associated with signs of behavioral impairment and intoxication. Given the important role that NMDA receptors play in synaptic transmission and brain function, it is important to understand the factors that regulate the ethanol inhibition of these receptors. One dynamic mechanism for regulating ethanol action may be via phosphorylation of NMDA subunits by serine–threonine and tyrosine kinases. Both NR1 and NR2 subunits contain multiple sites of phosphorylation; and in the NR1 subunit, most of these are contained within the C1 domain, a carboxy-terminal cassette that is subject to alternative splicing. Although results from our previous studies suggest that single phosphorylation sites do not greatly affect ethanol sensitivity of NMDA receptors, it is likely that in vivo, these subunits are phosphorylated at multiple sites by different kinases. In the present study, we constructed a series of NMDA receptor mutants at serine (S) or threonine (T) residues proposed to be sites of phosphorylation by protein kinase A and various isoforms of protein kinase C. Ethanol (100 mM) inhibited currents from wild-type NR1/2A and NR1/2B receptors expressed in human embryonic kidney 293 cells by approximately 25 and 30%, respectively. This inhibition was not different in single-site mutants expressing alanine (A) or aspartate/glutamate (D/E) at positions T879, S896, or T900. The mutant NR1(S890D) showed greater ethanol inhibition than NR1(890A) containing receptors, although this was only observed when it was combined with the NR2A subunit. Ethanol inhibition was not altered by aspartate substitution at four serines (positions 889, 890, 896, and 897) or when T879D was added to the four serine-substituted mutant. Ethanol inhibition was increased when T900E was added to the five serine-/threonine-substituted mutants, but again this was selective for NR2A containing receptors. Together with previously published data, these findings suggest that modification of putative phosphorylation sites could contribute to the overall acute ethanol sensitivity of recombinant NMDA receptors. Supported by R37AA009986.

Pregnenolone sulfate modulation of N-methyl-d-aspartate receptors is phosphorylation dependent

Pregnenolone sulfate (PS), an endogenously occurring neurosteroid, has been shown to modulate the activity of several neurotransmitter-gated channels, including the N-methyl-d-aspartate receptor (NMDAR). NMDARs are glutamate-gated ion channels involved in excitatory synaptic transmission, synaptic plasticity, and excitotoxicity. To determine the mechanism that controls PS sensitivity of NMDARs, we measured NMDAR responses induced by exogenous agonist application in voltage-clamped HEK293 cells expressing NR1/NR2B NMDARs and cultured rat hippocampal neurons. We report that PS potentiates the amplitude of whole-cell recorded NMDAR responses in cultured hippocampal neurons and HEK293 cells; however, the potentiating effect of PS on NMDAR in outside-out patches isolated from cultured hippocampal neurons and HEK293 cells was lost within 2 min after patch isolation in a neurosteroid-specific manner. The rate of diminution of the PS potentiating effect was slowed by protein phosphatase inhibitors. Treatment of cultured hippocampal neurons with a nonspecific protein kinase inhibitor and a specific protein kinase A (PKA) inhibitor diminished PS-induced potentiation, which was recovered by adding a PKA, but not a protein kinase C (PKC), activator. These results suggest that the effect of PS on NMDARs is controlled by cellular mechanisms that are mediated by dephosphorylation/phosphorylation pathways.

Structural Basis of NR2B-Selective Antagonist Recognition byN-Methyl-d-aspartate Receptors

N-Methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors endowed with unique pharmacological and functional properties. In particular, their high permeability to calcium ions confers on NMDARs a central role in triggering long term changes in synaptic strength. Under excitotoxic pathological conditions, such as those occurring during brain trauma, stroke, or Parkinson's or Huntington's diseases, calcium influx through NMDAR channels can also lead to neuronal injury. This argues for the use of NMDAR antagonists as potential therapeutic agents. To date, the most promising NMDAR antagonists are ifenprodil and derivatives, compounds that act as noncompetitive inhibitors selective for NMDARs containing the NR2B subunit. Recent studies have identified the large N-terminal domain (NTD) of NR2B as the region controlling ifenprodil sensitivity of NMDARs. We present here a detailed characterization of the ifenprodil binding site using both experimental and computational approaches. 3D homology modeling reveals that ifenprodil fits well in a closed cleft conformation of the NRB NTD; however, ifenprodil can adopt either of two possible binding orientations of opposite direction. By studying the effects of cleft mutations, we show that only the orientation in which the phenyl moiety points deep toward the NTD hinge is functionally relevant. Moreover, based on our model, we identify novel NTD NR2B residues that are crucial for conferring ifenprodil sensitivity and provide functional evidence that these residues directly interact with the ifenprodil molecule. This work provides a general insight into the origin of the subunit-selectivity of NMDAR noncompetitive antagonists and offer clues for the discovery of novel NR2B-selective antagonists.

Activation of the spinal sigma‐1 receptor enhances NMDA‐induced pain via PKC‐ and PKA‐dependent phosphorylation of the NR1 subunit in mice

Previously we demonstrated that the spinal sigma-1 receptor (Sig-1 R) plays an important role in pain transmission, although the exact mechanism is still unclear. It has been suggested that Sig-1 R agonists increase glutamate-induced calcium influx through N-methyl-D-aspartate (NMDA) receptors. Despite data suggesting a link between Sig-1 Rs and NMDA receptors, there are no studies addressing whether Sig-1 R activation directly affects NMDA receptor sensitivity. We studied the effect of intrathecal (i.t.) administration of Sig-1 R agonists on protein kinase C (PKC) and protein kinase A (PKA) dependent phosphorylation of the NMDA receptor subunit NR1 (pNR1) as a marker of NMDA receptor sensitization. In addition, we examined whether this Sig-1 R mediated phosphorylation of NR1 plays an important role in sensory function using a model of NMDA-induced pain. Both Western blot assays and image analysis of pNR1 immunohistochemical staining in the spinal cord indicated that i.t. injection of the Sig-1 R agonists, PRE-084 or carbetapentane dose dependently enhanced pNR1 expression in the murine dorsal horn. This increased pNR1 expression was significantly reduced by pretreatment with the specific Sig-1 R antagonist, BD-1047. In another set of experiments Sig-1 R agonists further potentiated NMDA-induced pain behaviour and pNR1 immunoreactivity and this was also reversed with BD-1047. The results of this study suggest that the activation of spinal Sig-1 R enhances NMDA-induced pain via PKC- and PKA-dependent phosphorylation of the NMDA receptor NR 1 subunit.

Ethanol inhibition of recombinant NMDA receptors is not altered by coexpression of CaMKII-α or CaMKII-β

Previous studies have shown that the N-methyl- d-aspartate (NMDA) receptor is an important target for the actions of ethanol in the brain. N-methyl- d-aspartate 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 postsynaptic 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 whereas 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 understanding 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. Calcium-calmodulin dependent protein kinase II is a major constituent of the postsynaptic density and is critically involved in various forms of learning and memory. NMDA receptor subunits were transiently expressed in human embryonic kidney 293 cells along with CaMKII-α or CaMKII-β tagged with the green fluorescent protein. Whole cell currents were elicited by brief exposures to glutamate and were measured using patch-clamp 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.

Enhanced Ethanol Inhibition of Recombinant N‐methyl‐D‐aspartate Receptors by Magnesium: Role of NR3A Subunits

The effects of ethanol on brain function are thought to be partly because of altered 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 influence 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. Recombinant cDNAs encoding NMDA receptor subunits were expressed in human embryonic kidney 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. 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. These results suggest that under physiological conditions, NR3A may be an important regulator of the acute ethanol sensitivity of brain NMDA receptors.

Bi-phasic intensity-dependent opioid-mediated neural amplitude changes in the chinchilla cochlea: Partial blockade by an N-Methyl- d-Aspartate (NMDA)-receptor antagonist

Dynorphins, glutamate, and glutamate-sensitive N-Methyl- d-Aspartate (NMDA) receptors exist in the mammalian cochlea. Dynorphins produce neural excitation and excitotoxic effects in the spinal cord through a κ-opioid facilitation of NMDA receptor-sensitivity to glutamate. The κ-opioid receptor drug agonists N-dimethylallyl-normetazocine [(−)-pentazocine (50 mmol)] and trans-3,4-dichloro- N-methyl- N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamide [U-50488H (100 mmol)] were administered across the cochlear round window membrane in the chinchilla. Each drug produced significant post-baseline amplitude changes in the click-evoked auditory nerve compound action potential. Amplitude changes at threshold amounted to increases in sensitivity that ranged from 4–8 decibels, measured in sound pressure level (dB SPL). The large neural amplitude increases at threshold were accompanied by progressively smaller amplitude changes at 5 and 10 dB above threshold (dB SL). However, at stimulus intensities ≥ 20 dB SL, post-baseline neural amplitudes were suppressed to levels below baseline and control values. These bi-phasic intensity-dependent neural amplitude changes have never before been observed following i.v. administered (−)-pentazocine in this species. Finally, the bi-phasic neural amplitude changes in U-50488H-treated (100 mmol) animals were partially blocked (except at 20 dB SL), following a round window pre-treatment with the NMDA receptor drug antagonist, dizocilpine hydrogen maleate [(+)-MK-801 (8 mmol)]. Our data suggests that endogenous dynorphins within lateral efferent olivocochlear neurons differentially modulate auditory neural excitation, possibly through cochlear NMDA receptors and glutamate. The role played by lateral efferent opioid neuromodulation at cochlear NMDA receptors, is discussed.

Potentiation of N-methyl- d-aspartate receptor-mediated neuronal injury during methamphetamine withdrawal in vitro requires co-activation of IP 3 receptors

Recent findings suggest that methamphetamine (METH) functions acutely to inhibit N-methyl- d-aspartate (NMDA) receptor function. Protracted withdrawal from METH exposure may increase the sensitivity of NMDA receptors to agonist exposure, promoting neuronal excitability. However, the relevance of METH effects on NMDA receptor activity with regard to neuronal viability has not been fully studied. The present studies examined the effects of protracted METH exposure (6 or 7 days; 1.0–100 μM) and withdrawal (1 or 7 days) on NMDA receptor-dependent neurotoxicity, determined with use of the non-vital fluorescent marker propidium iodide, in organotypic slice cultures of male and female rats. Prolonged exposure to METH (100 μM) produced only modest toxicity in the granule cell layer of the dentate gyrus. Withdrawal from METH exposure (1 or 7 days) did not produce overt neuronal injury in any region of slice cultures. Exposure to NMDA (5 μM) produced marked neurotoxicity in the CA1 pyramidal cell layer. Neither co-exposure to METH nor 1 day of METH withdrawal in combination with NMDA exposure altered NMDA-induced neurotoxicity. In contrast, protracted withdrawal from METH exposure (7 days) was associated with a marked (∼400%) increase in NMDA-induced neurotoxicity in CA1 region pyramidal cells. This potentiation of neurotoxicity was prevented by co-exposure to the selective NMDA receptor antagonist 5-2-amino-5-phoshonovaleric acid (20 μM) and was markedly attenuated by co-exposure of slices to xestospongin C (1 μM), an antagonist of IP 3 receptors. The results of the present studies suggest that long-term METH withdrawal functionally sensitizes the NMDA receptor to agonist exposure and requires the co-activation of NMDA and IP 3 receptors.

Increased Ethanol Resistance and Consumption in Eps8 Knockout Mice Correlates with Altered Actin Dynamics

Dynamic modulation of the actin cytoskeleton is critical for synaptic plasticity, abnormalities of which are thought to contribute to mental illness and addiction. Here we report that mice lacking Eps8, a regulator of actin dynamics, are resistant to some acute intoxicating effects of ethanol and show increased ethanol consumption. In the brain, the N-methyl-D-aspartate (NMDA) receptor is a major target of ethanol. We show that Eps8 is localized to postsynaptic structures and is part of the NMDA receptor complex. Moreover, in Eps8 null mice, NMDA receptor currents and their sensitivity to inhibition by ethanol are abnormal. In addition, Eps8 null neurons are resistant to the actin-remodeling activities of NMDA and ethanol. We propose that proper regulation of the actin cytoskeleton is a key determinant of cellular and behavioral responses to ethanol.

A Reconsideration of the Relevance of Systemic Low-Dose Ketamine to the Pathophysiology of Fibromyalgia

Fibromyalgia is a common disorder characterized by chronic widespread pain that affects an estimated 2% of the general population. Recent advances have shed insight on this mysterious disorder, leading to the growing conclusion that disturbances of pain-related processes within the central nervous system, termed central sensitization, represent its most likely source. The phenomenon of central sensitization depends on plasticity in function of N-methyl-D-aspartate (NMDA) subtype glutamate receptors. Earlier studies implicated increased sensitivity of central NMDA receptors as playing a primary role in fibromyalgia, as evidenced by a significant reduction in symptoms among a large subset of patients in response to low doses of ketamine, a noncompetitive NMDA receptor antagonist. However, recent insights into the pharmacology of this drug cast doubt on a direct contribution of NMDA receptors and add credence to a model of the disorder that suggests that the primary pathology of fibromyalgia is a suppression of the normal activity of dopamine-releasing neurons within the limbic system. The implications for future therapies for fibromyalgia, and indeed many other chronic pain conditions, are discussed in light of these insights. Perspective The current lack of a demonstrable pathology underlying the pain of fibromyalgia has hampered progress toward adequate treatment of this mysterious disorder. Accumulating evidence suggests that fibromyalgia may represent a dysregulation of dopaminergic neurotransmission, which may provide insights to guide both rational clinical interventions as well as system-specific research models.

Effects of 8 Different NR1 Splice Variants on the Ethanol Inhibition of Recombinant NMDA Receptors

N-Methyl-D-aspartate (NMDA) receptors are glutamate-activated ion channels that are assembled from NR1 and NR2 subunits. These receptors are highly enriched in brain neurons and are considered to be an important target for the acute and chronic effects of ethanol. NR2 subunits (A-D) arise from separate genes and are expressed in a developmental and brain region-specific manner. The NR1 subunit has 8 isoforms that are generated by alternative splicing of a single gene. The heteromeric subunit makeup of the NMDA receptor determines the pharmacological and biophysical properties of the receptor and provides for functional receptor heterogeneity. Although results from previous studies suggest that NR2 subunits affect the ethanol sensitivity of NMDA receptors, the role of the NR1 subunit and its multiple splice variants is less well known. In this study, all 8 NR1 splice variants were individually coexpressed with each NR2 subunit in human embryonic kidney 293 (HEK293) cells and tested for inhibition by ethanol using patch-clamp electrophysiology. All 32 subunit combinations tested gave reproducible glutamate-activated currents and all receptors were inhibited to some degree by 100 mM ethanol. The sensitivity of individual receptors to ethanol was affected by the specific NR1 splice variant expressed with receptors containing the NR1-3 and NR1-4 subunits among the least inhibited by ethanol. These results suggest that regional, developmental, or compensatory changes in the expression of NR1 splice variants may significantly affect ethanol inhibition of NMDA receptors.

Effects of Amino Acid Substitutions in Transmembrane Domains of the NR1 Subunit on the Ethanol Inhibition of Recombinant N‐Methyl‐d‐aspartate Receptors

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor is involved in a variety of processes that regulate neuronal plasticity and is an important target for the acute and chronic effects of ethanol. However, the specific sites where ethanol interacts with the receptor protein have yet to be fully elucidated. We previously demonstrated that a phenylalanine to alanine mutation in the third transmembrane domain (TM3) of the NR1 subunit decreased the ethanol inhibition of NMDA receptors expressed in HEK293 cells. In this study, we characterized the ethanol inhibition of NMDA receptors containing additional mutations within the TM3 and TM4 domains of the NR1 subunit. Site-directed mutagenesis was used to alter specific amino acid residues in the TM3 and TM4 domains of the NR1 subunit. Mutant NR1 subunits were coexpressed with the NR2A subunit in HEK293 cells and examined for alterations in ethanol sensitivity using whole-cell voltage-clamp electrophysiology. Replacing phenylalanine at TM3 position 639 in the NR1 subunit (F639) with 9 different amino acids produced functional receptors when coexpressed with the NR2A subunit. All mutants showed a concentration-dependent inhibition by ethanol (10-100 mM), with the alanine and serine mutants being significantly less sensitive to ethanol. Amino acid substitutions at the F639 site also produced variable changes in the concentration-response relationship to glycine. However, no significant correlation between glycine EC(50) values and the magnitude of ethanol inhibition was observed. Alanine mutations at TM4 positions 813 (M813A) and 819 (L819A), but not at 817 (F817A), of the NR1 subunit enhanced ethanol inhibition. Substitution of tryptophan for TM4 residues in the NR1 subunit (positions 820-822) that are homologous to a site in the NR2A subunit shown to reduce ethanol inhibition (A825W) had no effect on ethanol sensitivity. However, these NR1 TM4 tryptophan mutants restored the ethanol inhibition of the NR1 TM3 F639A mutant to wild-type levels in a stepwise fashion. These results indicate that the ethanol sensitivity of NMDA receptors may be regulated by discrete sites within the TM3 and TM4 domains of the NR1 subunit.

Neonatal Toluene Exposure Alters Agonist and Antagonist Sensitivity and NR2B Subunit Expression of NMDA Receptors in Cultured Cerebellar Granule Neurons

Toluene has been reported to antagonize the function of N-methyl-D-aspartate (NMDA) receptors. In this study, the effects of neonatal toluene exposure on NMDA receptors in primarily cultured cerebellar granule neurons were examined. Sprague-Dawley rats were treated with toluene (0, 200, 500, and 1000 mg/kg, i.p.) from postnatal day (PN) 4 to PN 7. Under toluene-free conditions, Ca2+ signals of cultured neurons in response to glutamate and NMDA were measured for up to 14 days. The expression of NMDA receptor subunits (NR1, NR2A, and NR2B) at 5-14 days in vitro (DIV) were also determined. Neonatal toluene exposure dose-dependently reduced intracellular Ca2+ signals in response to glutamate/glycine and NMDA/glycine in cultured cerebellar granule neurons, and these effects were gradually decreased with time. Such toluene exposure did not influence the inhibition of Mg2+ or MK801 on NMDA-evoked responses, but it decreased the potency of ifenprodil (an NR2B preferring antagonist). The protein levels of NMDA receptor subunit NR2B were consistently reduced by toluene exposure at 5 DIV, but not at 14 DIV. These results demonstrate that neonatal toluene exposure induces long-term but reversible changes in the function and composition of NMDA receptors. Such changes during developmental stages may contribute to the cerebellar dysfunction observed in fetal solvent syndrome.

Differential Effects on Current Kinetics by Point Mutations in the lurcher Motif of NR1/NR2A Receptors

The extracellular proton site has emerged as the key site in regulating N-methyl-d-aspartate (NMDA) receptor function. However, the molecular determinants for the proton-sensitive gating of NMDA receptors are still not clearly delineated. The highly conserved lurcher motif plays an important role in determining the proton sensitivity of NMDA receptors. Mutations of several residues in the lurcher motif of either NR1 or NR2A significantly reduce proton sensitivity of recombinant NR1/NR2A receptors. It remains uncertain how these residues play a role in proton inhibition of NMDA receptors. Mutations of these residues could directly reduce the proton affinity. Alternatively, they could alter the proton IC(50) indirectly by increasing channel open probability. In the present study, we recorded the macroscopic NMDA currents in HEK 293 cells with a piezo-based rapid solution exchange system. We show that zinc slows the deactivation of NR1a(A653T)/NR2A receptors and NR1a/NR2A(A651T) receptors. However, NR1a(T648C)/NR2A, NR1a/NR2A(T646C), NR1a(A649C)/NR2A, NR1a/NR2A(A647C), and NR1a(A653T)/NR2A exhibit significantly slower rise time and deactivation time constants under nominally zinc-free conditions. Our data suggest that the channel open probability for these mutant receptors may be significantly increased. The reduction in proton sensitivity by these mutations could be accounted for, at least partially, by the increased channel open probability. In contrast, NR1a/NR2A(A651T) exhibits normal macroscopic currents, suggesting that the reduction of proton sensitivity by this mutation cannot be attributed to any significant change of open probability. Further experiments are needed to determine the exact role of this residue in proton-sensitive gating of NMDA receptors.

Fyn kinase does not reduce ethanol inhibition of zinc-insensitive NR2A-containing N-methyl- d-aspartate receptors

N-methyl- d-aspartate (NMDA) receptors are ion channels activated by the neurotransmitter glutamate and are important mediators of neuronal signal transduction. Ethanol inhibits ion flux through NMDA receptors at concentrations that are associated with behavioral signs of intoxication. The overall sensitivity of NMDA receptors to ethanol is influenced by factors, including subunit composition and interactions with cytoskeletal elements. Results of studies also support the suggestion that the ethanol inhibition on NR1/2A receptors is reduced by Fyn kinase–mediated tyrosine phosphorylation. However, tyrosine kinases also reduce the high-affinity zinc sensitivity of NR1/2A receptors, supporting the suggestion that kinase-dependent effects on ethanol inhibition may be secondary to relief of zinc inhibition. In the current study, the effect of Fyn kinase on the ethanol inhibition of NR1/2A receptors was determined under conditions in which zinc sensitivity is eliminated. Human embryonic kidney 293 (HEK 293) cells were transiently transfected with wild-type or mutant NMDA subunits, and glutamate-activated currents were measured by using patch-clamp electrophysiology. Inclusion of the tyrosine phosphatase inhibitor potassium bisperoxo(1,10-phenanthroline)oxovanadate (V) [bpV(phen)] in the recording pipette eliminated the potentiation of NR1/2A currents by heavy metal chelators. Under these conditions, Fyn kinase did not reduce ethanol inhibition of wild-type receptors. Fyn kinase also had no effect on the magnitude of ethanol inhibition of zinc-insensitive NR1/2A(H128S) receptors. Together, results of the current study indicate that Fyn kinase does not directly affect the ethanol sensitivity of NR1/2A receptors.