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 (100 mM) and toluene (1 or 3 mM) 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.

A novel substituted aminoquinoline selectively targets voltage-sensitive sodium channel isoforms and NMDA receptor subtypes and alleviates chronic inflammatory and neuropathic pain.

Recent understanding of the systems that mediate complex disease states, has generated a search for molecules that simultaneously modulate more than one component of a pathologic pathway. Chronic pain syndromes are etiologically connected to functional changes (sensitization) in both peripheral sensory neurons and in the central nervous system (CNS). These functional changes involve modifications of a significant number of components of signal generating, signal transducing and signal propagating pathways. Our analysis of disease-related changes which take place in sensory neurons during sensitization led to the design of a molecule that would simultaneously inhibit peripheral NMDA receptors and voltage sensitive sodium channels. In the current report, we detail the selectivity of N,N-(diphenyl)-4-ureido-5,7-dichloro-2-carboxy-quinoline (DCUKA) for action at NMDA receptors composed of different subunit combinations and voltage sensitive sodium channels having different α subunits. We show that DCUKA is restricted to the periphery after oral administration, and that circulating blood levels are compatible with its necessary concentrations for effects at the peripheral cognate receptors/channels that were assayed in vitro. Our results demonstrate that DCUKA, at concentrations circulating in the blood after oral administration, can modulate systems which are upregulated during peripheral sensitization, and are important for generating and conducting pain information to the CNS. Furthermore, we demonstrate that DCUKA ameliorates the hyperalgesia of chronic pain without affecting normal pain responses in neuropathic and inflammation-induced chronic pain models.

Altered NMDA receptor function in primary cultures of hippocampal neurons from mice lacking the Homer2 gene.

N-Methyl-D-Aspartate (NMDA) receptors are inhibited during acute exposure to ethanol and are involved in changes in neuronal plasticity following repeated ethanol exposure. The postsynaptic scaffolding protein Homer2 can regulate the cell surface expression of NMDA receptors in vivo, and mice with a null mutation of the Homer2 gene exhibit an alcohol-avoiding and -intolerant phenotype that is accompanied by a lack of ethanol-induced glutamate sensitization. Thus, Homer2 deletion may perturb the function or acute ethanol sensitivity of the NMDA receptor. In this study, the function and ethanol sensitivity of glutamate receptors in cultured hippocampal neurons from wild-type (WT) and Homer2 knock-out (KO) mice were examined at 7 and 14 days in vitro (DIV) using standard whole-cell voltage-clamp electrophysiology. As compared with wild-type controls, NMDA receptor current density was reduced in cultured hippocampal neurons from Homer2 KO mice at 14 DIV, but not at 7 DIV. There were no genotype-dependent changes in whole-cell capacitance or in currents evoked by kainic acid. The GluN2B-selective antagonist ifenprodil inhibited NMDA-evoked currents to a similar extent in both wild-type and Homer2 KO neurons and inhibition was greater at 7 versus 14 DIV. NMDA receptor currents from both WT and KO mice were inhibited by ethanol (10-100 mM) and the degree of inhibition did not differ as a function of genotype. In conclusion, NMDA receptor function, but not ethanol sensitivity, is reduced in hippocampal neurons lacking the Homer2 gene.

Cysteine substitution of transmembrane domain amino acids alters the ethanol inhibition of GluN1/GluN2A N-methyl-D-aspartate receptors.

N-Methyl-d-aspartate receptors (NMDARs) are inhibited by behaviorally relevant concentrations of ethanol, and residues within transmembrane (TM) domains of NMDARs, including TM3 GluN1 phenylalanine 639 (F639), regulate this sensitivity. In the present study, we used cysteine (C) mutagenesis to determine whether there are additional residues within nearby TM domains that regulate ethanol inhibition on NMDARs. GluN1(F639C)/GluN2A receptors were less inhibited by ethanol than wild-type receptors, and inhibition was restored to wild-type levels following treatment with ethanol-like methanethiosulfonate reagents. Molecular modeling identified six residues in the GluN1 TM1 domain (valine V566; serine S569) and the GluN2A TM4 domain (methionine, M817; V820, F821, and leucine, L824) that were in close vicinity to the TM3 F639 residue, and these were individually mutated to cysteine and tested for ethanol inhibition and receptor function. The F639C-induced decrease in ethanol inhibition was blunted by coexpression of GluN1 TM1 mutants V566C and S569C, and statistically significant interactions were observed for ethanol inhibition among V566C, F639C, and GluN2A TM4 mutants V820C and F821C and S569C, F639C, and GluN2A TM4 mutants F821C and L824C. Ethanol inhibition was also reduced when either GluN1 TM1 mutant V566C or S569C was combined with GluN2A V820C, suggesting a novel TM1:TM4 intrasubunit site of action for ethanol. Cysteines substituted at TM3 and TM4 sites previously suggested to interact with ethanol had less dramatic effects on ethanol inhibition. Overall, the results from these studies suggest that interactions among TM1, TM3, and TM4 amino acids in NMDARs are important determinants of ethanol action at these receptors.

Dephosphorylation of GluN2B C-terminal tyrosine residues does not contribute to acute ethanol inhibition of recombinant NMDA receptors.

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.

Ethanol inhibition of constitutively open N-methyl-D-aspartate receptors.

N-Methyl-D-aspartate (NMDA) receptors gate a slow and calcium-rich component of the postsynaptic glutamate response. Like all ionotropic glutamate receptors, NMDA subunits contain a highly conserved motif (SYTANLAAF) in the transmembrane (TM) 3 domain that is critically involved in channel gating. Mutation of an alanine in this domain (A7; underlined above) results in constitutively open receptors that show reduced sensitivity to several allosteric modulators. In this study, we examined the effects of ethanol, a substance that inhibits NMDA currents via an unknown mechanism, on tonically active NMDA receptors expressed in human embryonic kidney 293 cells. Ethanol (100 mM) inhibited currents from GluN1(A7R)/GluN2A and GluN1(A7R)/GluN2B receptors by approximately 50%, whereas those from GluN1/GluN2B(A7R) receptors were reduced by less than 10%. In cysteine-substituted GluN1 and GluN2 A7 mutants, estimated ethanol IC50 values for agonist-gated currents were 101, 117, 103, and 69 mM for GluN1(A7C)/GluN2A, GluN1(A7C)/GluN2B, GluN1/GluN2A(A7C), and GluN1/GluN2B(A7C) receptors, respectively. After exposure to the thiol-modifying reagent 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET), A7C mutants showed robust agonist-independent currents and reduced sensitivity to ethanol (IC50 values of 371, 256, 715, and 958 mM, respectively, as above). In contrast, cysteine modification of the ligand-binding domain resulted in constitutively open receptors that showed robust ethanol inhibition. Ethanol inhibition of MTSET-treated GluN1(A7C) receptors was further reduced by TM3/TM4 mutations previously shown to reduce ethanol sensitivity of agonist-gated receptors. Overall, these results show that ethanol affects NMDA receptor function at a site distal from agonist binding and appears to exert greater effects via perturbation of GluN2 subunits.

Expression of glycine-activated diheteromeric NR1/NR3 receptors in human embryonic kidney 293 cells Is NR1 splice variant-dependent.

In oocytes, glycine activates receptors formed by diheteromeric combinations of N-methyl-d-aspartate (NMDA) NR1 and NR3 subunits. In contrast, functional receptors in mammalian cells require the simultaneous expression of NR1 and both NR3A and NR3B subunits. In vivo, NR3A and NR3B subunits show differential expression patterns and thus may not naturally form triheteromeric receptors. In this study, we examined whether NR1 splice variants play a role in allowing assembly of functional diheteromeric receptors in mammalian cells. Little current was found in human embryonic kidney 293 cells coexpressing either NR3A or NR3B and the NR1-1a splice variant. However, robust glycine-activated currents were generated in cells transfected with NR3(A or B) and either NR1-2a, NR1-3a, or NR1-4a, and current density was correlated with NR1 C-terminal length. Truncation of the NR1-1a C terminus modestly enhanced NR1-1a/NR3A currents, whereas only small increases were observed with mutations of C-terminal residues that control trafficking or phosphorylation. In contrast, large currents were observed when an extracellular phenylalanine in NR1-1a that influences glycine access was mutated to alanine. A separate mutation in NR1-1a that disrupts glycine binding did not generate responses in NR1-1a/NR3A receptors alone, but it produced a greater than 30-fold potentiation of currents during coapplication of glycine and the glycine antagonist 7-chlorokynurenic acid. Finally, transfection of cells with the NR1-4a subunit along with NR2 and NR3 subunits resulted in the expression of both NR1/NR3 receptors and conventional NMDA receptor currents. These results indicate a prominent role for NR1 splice variants in the functional expression of NR1/NR3 receptors in mammalian cells.

Interferon-alpha causes neuronal dysfunction in encephalitis.

Interferon-alpha (IFNalpha) is a pleomorphic cytokine produced by nucleated cells in response to viral infection. In patients, treatment with IFNalpha has side effects including cognitive impairment resembling subcortical dementia, which is a hallmark of human immunodeficiency virus (HIV)-associated dementia (HAD). IFNalpha is increased in the CSF of HAD patients compared with HIV patients without dementia. In this study, blocking IFNalpha in a HIV encephalitis (HIVE) mouse model with intraperitoneal injections of IFNalpha neutralizing antibodies (NAbs) significantly improved cognitive function compared with untreated or control antibody-treated HIVE mice during water radial arm maze behavioral testing. Treatment with IFNalpha NAbs significantly decreased microgliosis and prevented loss of dendritic arborization in the brains of HIVE mice. Furthermore, treatment of primary neuron cultures with IFNalpha resulted in dose-dependent loss of dendritic arborization that was blocked with IFNalpha NAb treatment and partially blocked with NMDA antagonists [AP5 and MK801 (dizocilpine maleate)] indicating glutamate signaling is involved in IFNalpha-mediated neuronal damage. These results show that IFNalpha has a major role in the pathogenesis of HIVE in mice and is likely important in the development neurocognitive dysfunction in humans with HIV. Blocking IFNalpha could be important in improving cognitive and pathological developments in HAD patients and may be clinically important in other neuroinflammatory diseases as well.

Enhanced ethanol inhibition of recombinant N-methyl-D-aspartate receptors by magnesium: role of NR3A subunits.

BACKGROUND: 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. METHODS: 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. 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.

Pharmacological characterization of glycine-activated currents in HEK 293 cells expressing N-methyl-D-aspartate NR1 and NR3 subunits.

N-Methyl-D-aspartate (NMDA) receptors are important targets for drugs of abuse such as ethanol, toluene, and ketamine. Ligand-gated ion channels assembled from the NR1 and NR3 subunits have functional and pharmacological properties that are distinct from those of conventional NMDA receptors containing NR2 subunits. In the present study we used voltage-clamp electrophysiology to characterize excitatory glycine-activated receptors assembled from NR1, NR3A, and NR3B subunits expressed in human embryonic kidney (HEK) 293 cells. These glycine-activated receptors were not stimulated by glutamate or kainic acid and were resistant to magnesium block. A wide variety of NMDA receptor antagonists including d-2-amino-5-phosphonovaleric acid, ifenprodil, memantine, (5R,10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclo-hepten-5,10-imine hydrogen maleate (MK-801) or acamprosate did not inhibit glycine-activated NR1/NR3A/NR3B receptors. Likewise, these receptors were not affected by antagonists of inhibitory glycine receptors or glycine transporters. The NMDA receptor glycine site agonist, d-serine, partially activated NR1/NR3A/NR3B receptors, whereas the antagonist, 5,7-dichloro-kynurenic acid, inhibited receptor currents. Conversely, the antagonist, 7-chlorokynurenic acid, and the partial agonist, R-(+)-3-amino-1-hydroxy-2-pyrrolidinone (HA-966), potentiated glycine-stimulated currents of these receptors. NR1/NR3A/NR3B receptor currents were inhibited by 10 to 21% by ethanol and toluene but were relatively insensitive to ketamine. Ethanol inhibition was enhanced in receptors expressing the NR1(L819A) mutant, whereas those containing NR1(F639A) or NR1(M813A) showed no change relative to the wild-type NR1. The results of this study indicate that coexpression of NR1, NR3A, and NR3B subunits in HEK 293 cells results in glycineactivated receptors with novel functional and pharmacological properties.

Effects of anesthetics on mutant N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

Alcohols, inhaled anesthetics, and some injectable anesthetics inhibit the function of N-methyl-d-aspartate (NMDA) receptors, but the mechanisms responsible for this inhibition are not fully understood. Recently, it was shown that ethanol inhibition of NMDA receptors was reduced by mutation of residues in the transmembrane (TM) segment 3 of the NR1 subunit (F639A) or in TM4 of the NR2A subunit (A825W), suggesting putative ethanol binding sites. We hypothesized that the actions of other anesthetics might also require these amino acids and evaluated the effects of anesthetics on the NMDA receptors expressed in Xenopus oocytes with two-electrode voltage-clamp recording. Effects of hexanol, octanol, isoflurane, halothane, chloroform, cyclopropane, 1-chloro-1,2,2-trifluorocyclobutane, and xenon were reduced or eliminated in the mutant NMDA receptors, whereas the inhibitory effects of nitrous oxide, ketamine, and benzene were not affected by these mutations. Rapid applications of glutamate and glycine by a T-tube device provided activation time constants, which suggested different properties of ketamine and isoflurane inhibition. Thus, amino acids in TM3 and TM4 are important for the actions of many anesthetics, but nitrous oxide, benzene, and ketamine seem to have distinct mechanisms for inhibition of the 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.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Effect of the NR3 subunit on ethanol inhibition of recombinant NMDA receptors.

N-Methyl-D-aspartate (NMDA) receptors are a subtype of glutamate receptor that serve important functions at glutamatergic synapses in the brain. NMDA receptors are inhibited by concentrations of ethanol that are associated with intoxication and chronic exposure of neurons to ethanol enhances NMDA receptor function. The factors that underlie the acute inhibition of NMDA receptors by ethanol are not completely known, but ethanol sensitivity is influenced by receptor subunit composition. In this study, the effect of the regulatory subunit, NR3, on ethanol inhibition of NMDA receptors was examined. Recombinant NMDA receptors comprised of NR1 and NR2 (A-D) subunits were transiently transfected into HEK293 cells in the absence or presence of the NR3 subunit. In the absence of NR3, all NMDA receptor subunit combinations were inhibited by 100 mM ethanol. Co-expression of NR3 or an NR3-GFP fusion protein with NR1/NR2 (A-D) subunits did not alter the inhibitory effects of ethanol. In addition, the inhibition of NR1/NR2B receptors by the NR2B subunit-selective antagonist, ifenprodil, was not altered by co-expression of the NR3 subunit. Overall, these results suggest that the NR3A subunit is not a determinant of ethanol sensitivity in recombinant NMDA receptors.