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ABSTRACT: Many studies examine the actions of ethanol on N-methyl-D-aspartate (NMDA) receptors using concentrations that are highly toxic (>or=100 mM). This study re-assesses the actions of ethanol at concentrations based around the US/UK 'drink-drive' limit (17 mM). Using two-electrode voltage-clamp recordings we examined the actions of ethanol on recombinant GluN1/GluN2A and GluN1/GluN2B NMDA receptors expressed in Xenopus laevis oocytes. We also investigated its actions on NMDA receptors containing GluN2A subunits with truncated or deleted carboxy terminal domains. Ethanol inhibition was voltage-independent and for GluN1/GluN2A NMDA receptors mean inhibition (20 mM at -60 mV) was 9.5+/-0.8% (n=33) while corresponding values for GluN1/GluN2B NMDA receptors were 6.5+/-0.8% (n=21). EC(50) values for glutamate at GluN1/GluN2A and glutamate and glycine at GluN1/GluN2B NMDA receptors were unaffected by the presence of ethanol. We did however observe a small increase in glycine potency, in the presence of ethanol, at GluN1/GluN2A NMDA receptors. Neither voltage-dependent Mg(2+) block nor memantine block was affected by ethanol. Reduced ethanol inhibition was observed however at NMDA receptors containing GluN2A subunits with mutated carboxy terminal domains. We conclude that the levels of inhibition seen with ethanol concentrations near to the US/UK drink-driving limit are very modest and even at higher (intoxicating) concentrations do not alter characteristic NMDA receptor properties.
European journal of pharmacology 04/2009; 614(1-3):14-21. · 2.59 Impact Factor
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Philip E Chen,
Michael L Errington,
Matthias Kneussel,
Guiquan Chen,
Alexander J Annala,
York H Rudhard,
Georg F Rast,
Christian G Specht,
Cezar M Tigaret,
Mohammed A Nassar,
Richard G M Morris,
Timothy V P Bliss,
Ralf Schoepfer
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ABSTRACT: The NMDA receptor (NMDAR) subunit GluN1 is an obligatory component of NMDARs without a known functional homolog and is expressed in almost every neuronal cell type. The NMDAR system is a coincidence detector with critical roles in spatial learning and synaptic plasticity. Its coincidence detection property is crucial for the induction of hippocampal long-term potentiation (LTP). We have generated a mutant mouse model expressing a hypomorph of the Grin1(N598R) allele, which leads to a minority (about 10%) of coincidence detection-impaired NMDARs. Surprisingly, these animals revealed specific functional changes in the dentate gyrus (DG) of the hippocampal formation. Early LTP was expressed normally in area CA1 in vivo, but was completely suppressed at perforant path-granule cell synapses in the DG. In addition, there was a pronounced reduction in the amplitude of the evoked population spike in the DG. These specific changes were accompanied by behavioral impairments in spatial recognition, spatial learning, reversal learning, and retention. Our data show that minor changes in GluN1-dependent NMDAR physiology can cause dramatic consequences in synaptic signaling in a subregion-specific fashion despite the nonredundant nature of the GluN1 gene and its global expression.
Learning & memory (Cold Spring Harbor, N.Y.) 01/2009; 16(10):635-44. · 4.08 Impact Factor
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Philip E Chen,
Matthew T Geballe,
Elyse Katz,
Kevin Erreger,
Matthew R Livesey,
Kate K O'Toole,
Phuong Le,
C Justin Lee,
James P Snyder,
Stephen F Traynelis,
David J A Wyllie
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ABSTRACT: Heteromeric NMDARs are composed of coagonist glycine-binding NR1 subunits and glutamate-binding NR2 subunits. The majority of functional NMDARs in the mammalian central nervous system (CNS) contain two NR1 subunits and two NR2 subunits of which there are four types (A-D). We show that the potency of a variety of endogenous and synthetic glycine-site coagonists varies between recombinant NMDARs such that the highest potency is seen at NR2D-containing and the lowest at NR2A-containing NMDARs. This heterogeneity is specified by the particular NR2 subunit within the NMDAR complex since the glycine-binding NR1 subunit is common to all NMDARs investigated. To identify the molecular determinants responsible for this heterogeneity, we generated chimeric NR2A/2D subunits where we exchanged the S1 and S2 regions that form the ligand-binding domains and coexpressed these with NR1 subunits in Xenopus laevis oocytes. Glycine concentration-response curves for NMDARs containing NR2A subunits including the NR2D S1 region gave mean glycine EC(50) values similar to NR2A(WT)-containing NMDARs. However, receptors containing NR2A subunits including the NR2D S2 region or both NR2D S1 and S2 regions gave glycine potencies similar to those seen in NR2D(WT)-containing NMDARs. In particular, two residues in the S2 region of the NR2A subunit (Lys719 and Tyr735) when mutated to the corresponding residues found in the NR2D subunit influence glycine potency. We conclude that the variation in glycine potency is caused by interactions between the NR1 and NR2 ligand-binding domains that occur following agonist binding and which may be involved in the initial conformation changes that determine channel gating.
The Journal of Physiology 02/2008; 586(1):227-45. · 4.72 Impact Factor
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ABSTRACT: N-methyl-d-aspartate receptors (NMDARs) display differences in their sensitivity to the channel blockers Mg(2+) and memantine that are dependent on the identity of the NR2 subunit present in the receptor-channel complex. This study used two-electrode voltage-clamp recordings from Xenopus laevis oocytes expressing recombinant NMDARs to investigate the actions of Mg(2+) and memantine at the two NMDARs displaying the largest differences in sensitivity to these blockers, namely NR1/NR2A and NR1/NR2D NMDARs. In addition, NR2A/2D chimeric subunits have been employed to examine the effects of pore-forming elements and ligand-binding domains (LBD) on the potency of the block produced by each of these inhibitors. Our results show that, as previously documented, NR2D-containing NMDARs are less sensitive to voltage-dependent Mg(2+) block than their NR2A-containing counterparts. The reduced sensitivity is determined by the M1M2M3 membrane-associated regions, as replacing these regions in NR2A subunits with those found in NR2D subunits results in a approximately 10-fold reduction in Mg(2+) potency. Intriguingly, replacing the NR2A LBD with that from NR2D subunits results in a approximately 2-fold increase in Mg(2+) potency. Moreover, when responses mediated by NR1/NR2A NMDARs are evoked by the partial agonist homoquinolinate, rather than glutamate, Mg(2+) also displays an increased potency. Memantine block of glutamate-evoked currents is most potent at NR1/NR2D NMDARs, but no differences are observed in its ability to inhibit NR2A-containing or NR2A/2D chimeric NMDARs. We suggest that the potency of block of NMDARs by Mg(2+) is influenced not only by pore-forming regions but also the LBD and the resulting conformational changes that occur following agonist binding.
The Journal of Physiology 02/2008; 586(1):211-25. · 4.72 Impact Factor
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Kevin Erreger,
Matthew T Geballe,
Anders Kristensen, Philip E Chen,
Kasper B Hansen,
C Justin Lee,
Hongjie Yuan,
Phuong Le,
Polina N Lyuboslavsky,
Nicola Micale,
Lars Jørgensen,
Rasmus P Clausen,
David J A Wyllie,
James P Snyder,
Stephen F Traynelis
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ABSTRACT: The four N-methyl-d-aspartate (NMDA) receptor NR2 subunits (NR2A-D) have different developmental, anatomical, and functional profiles that allow them to serve different roles in normal and neuropathological situations. Identification of subunit-selective NMDA receptor agonists, antagonists, or modulators could prove to be both valuable pharmacological tools as well as potential new therapeutic agents. We evaluated the potency and efficacy of a wide range of glutamate-like compounds at NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D receptors. Twenty-five of 53 compounds examined exhibited agonist activity at the glutamate binding site of NMDA receptors. Concentration-response relationships were determined for these agonists at each NR2 subunit. We find consistently higher potency at the NR2D subunit for a wide range of dissimilar structures, with (2S,4R)-4-methylglutamate (SYM2081) showing the greatest differential potency between NR2A- and NR2D-containing receptors (46-fold). Analysis of chimeric NR2A/D receptors suggests that enhanced agonist potency for NR2D is controlled by residues in both of the domains (Domain1 and Domain2) that compose the bilobed agonist binding domain. Molecular dynamics (MD) simulations comparing a crystallography-based hydrated NR1/NR2A model with a homology-based NR1/NR2D hydrated model of the agonist binding domains suggest that glutamate exhibits a different binding mode in NR2D compared with NR2A that accommodates a 4-methyl substitution in SYM2081. Mutagenesis of functionally divergent residues supports the conclusions drawn based on the modeling studies. Despite high homology and conserved atomic contact residues within the agonist binding pocket of NR2A and NR2D, glutamate adopts a different binding orientation that could be exploited for the development of subunit selective agonists and competitive antagonists.
Molecular Pharmacology 11/2007; 72(4):907-20. · 4.88 Impact Factor
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ABSTRACT: Mice carrying the spontaneous genetic mutation known as Wallerian degeneration slow (Wlds) have a unique neuroprotective phenotype, where axonal and synaptic compartments of neurons are protected from degeneration following a wide variety of physical, toxic and inherited disease-inducing stimuli. This remarkable phenotype has been shown to delay onset and progression in several mouse models of neurodegenerative disease, suggesting that Wlds-mediated neuroprotection may assist in the identification of novel therapeutic targets. As a result, cross-breeding of Wlds mice with mouse models of neurodegenerative diseases is used increasingly to understand the roles of axon and synapse degeneration in disease. However, the phenotype shows strong gene-dose dependence so it is important to distinguish offspring that are homozygous or heterozygous for the mutation. Since the Wlds mutation comprises a triplication of a region already present in the mouse genome, the most stringent way to quantify the number of mutant Wlds alleles is using copy number. Current approaches to genotype Wlds mice are based on either Southern blots or pulsed field gel electrophoresis, neither of which are as rapid or efficient as quantitative PCR (QPCR).
We have developed a rapid, robust and efficient genotyping method for Wlds using QPCR. This approach differentiates, based on copy number, homozygous and heterozygous Wlds mice from wild-type mice and each other. We show that this approach can be used to genotype mice carrying the spontaneous Wlds mutation as well as animals expressing the Wlds transgene.
We have developed a QPCR genotyping method that permits rapid and effective genotyping of Wlds copy number. This technique will be of particular benefit in studies where Wlds mice are cross-bred with other mouse models of neurodegenerative disease in order to understand the neuroprotective processes conferred by the Wlds mutation.
Molecular Neurodegeneration 02/2007; 2:21. · 4.28 Impact Factor
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ABSTRACT: We have quantified the effects of the N-methyl-d-aspartate (NMDA) receptor antagonist (R)-[(S)-1-(4-bromo-phenyl)-ethylamino]-(2,3-dioxo-1,2,3,4-tetrahydroquinoxalin-5-yl)-methyl]-phosphonic acid (NVP-AAM077) at rat recombinant N-methyl-D-aspartate receptor (NR)1/NR2A and NR1/NR2B NMDA receptors expressed in Xenopus laevis oocytes. We observed no difference in the steady-state levels of inhibition produced by NVP-AAM077 when it was either preapplied or coapplied with glutamate. The IC50 values for NVP-AAM077 acting at NR1/NR2A NMDA receptors were, as expected, dependent on the glutamate concentration used to evoke responses, being 31 +/- 2 nM (with glutamate at its EC50 concentration) and 214 +/- 10 nM (at 10 times the EC50 concentration). Schild analysis confirmed that the antagonism produced by NVP-AAM077 at NR1/NR2A NMDA receptors was competitive and gave an estimate of its equilibrium constant (K(B)) of 15 +/- 2 nM. Furthermore, Schild analysis of an NMDA receptor carrying a threonine-to-alanine point mutation in the NR2A ligand binding site indicated that NVP-AAM077 still acted in a competitive manner but with its K(B) increased by around 15-fold. At NR1/NR2B NMDA receptors, NVP-AAM077 displayed reduced potency. An IC50 value of 215 +/- 13 nM was obtained in the presence of the EC50 concentration of glutamate (1.5 microM), whereas a value of 2.2 +/- 0.14 microM was obtained with higher (15 microM) glutamate concentrations. Schild analysis gave a K(B) for NVP-AAM077 at NR2B-containing receptors of 78 +/- 3 nM. Finally, using a kinetic scheme to model "synaptic-like" activation of NMDA receptors, we show that the difference in the equilibrium constants for NVP-AAM077 is not sufficient to discriminate between NR2A-containing or NR2B-containing NMDA receptors.
Molecular Pharmacology 10/2006; 70(3):1022-32. · 4.88 Impact Factor
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ABSTRACT: We have examined the function of a conserved serine residue (Ser670) in the S2 ligand-binding region of the NR2A N-methyl-d-aspartate (NMDA) receptor subunit, using recombinant NR1/NR2A receptors expressed in Xenopus laevis oocytes. Mutation of Ser670 to glycine (S670G) in NR2A reduced the potency of glutamate by 124-fold. Single-channel conductance and the duration of apparent open periods of NR2A(S670G) receptor mutants were, however, indistinguishable from wild-type NMDA receptors. NR1/NR2A(S670G) shut-time distributions were best described by a mixture of six exponential components, and the four shortest shut intervals of each distribution were considered to occur within a channel activation (burst). Bursts of single-channel openings were fitted with a mixture of four exponential components. The longest two components carried the majority of the charge transfer and had mean durations of 9.6 +/- 0.5 and 29.6 +/- 1.5 ms. The overall channel open probability during a burst was high (mean, 0.83 +/- 0.06). Consistent with a shortening of NMDA receptor-channel burst lengths was the observation of an increased deactivation rate of macroscopic currents evoked by brief applications of glutamate to outside-out membrane patches. Correlations between shut times and adjacent open times were observed in all data records. Noticeably, shorter than average openings tended to occur next to long closed periods, whereas longer than average openings tended to occur next to short closings. Our single-channel data, together with modelling using a kinetic scheme to describe channel activations, support our hypothesis that the S670G point mutation reduces the dwell time of glutamate in its binding site.
The Journal of Physiology 08/2006; 574(Pt 2):477-89. · 4.72 Impact Factor
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ABSTRACT: Ionotropic glutamate receptors mediate the vast majority of fast excitatory synaptic transmission in the CNS. Elucidating the structure of these proteins is central to understanding their overall function and in the last few years a tremendous amount of knowledge has been gained from the crystal structures of the ligand-binding domains of the receptor protein. These efforts have enabled us to unravel the possible mechanisms of partial agonism, agonist selectivity and desensitization. This review summarizes recent data obtained from structural studies of the binding pockets of the GluR2, GluR5/6, NR1 and NR2A subunits and discusses these studies together with homology modelling and molecular dynamics simulations that have suggested possible binding modes for full and partial agonists as well as antagonists within the binding pocket of various ionotropic glutamate receptor subunits. Comparison of the ligand-binding pockets suggests that the ligand-binding mechanisms may be conserved throughout the glutamate receptor family, although agonist selectivity may be explained by a number of features inherent to the AMPA, kainate and NMDA receptor-binding pockets such as steric occlusion, cavity size and the contribution of water-bridged interactions.
British Journal of Pharmacology 05/2006; 147(8):839-53. · 4.41 Impact Factor
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Thomas H Gillingwater,
Thomas M Wishart, Philip E Chen,
Jane E Haley,
Kevin Robertson,
Stephen H-F MacDonald,
Susan Middleton,
Kolja Wawrowski,
Michael J Shipston,
Shlomo Melmed,
David J A Wyllie,
Paul A Skehel,
Michael P Coleman,
Richard R Ribchester
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ABSTRACT: Wallerian degeneration of injured neuronal axons and synapses is blocked in Wld(S) mutant mice by expression of an nicotinamide mononucleotide adenylyl transferase 1 (Nmnat-1)/truncated-Ube4b chimeric gene. The protein product of the Wld(S) gene localizes to neuronal nuclei. Here we show that Wld(S) protein expression selectively alters mRNA levels of other genes in Wld(S) mouse cerebellum in vivo and following transfection of human embryonic kidney (HEK293) cells in vitro. The largest changes, identified by microarray analysis and quantitative real-time polymerase chain reaction of cerebellar mRNA, were an approximate 10-fold down-regulation of pituitary tumour-transforming gene-1 (pttg1) and an approximate 5-fold up-regulation of a structural homologue of erythroid differentiation regulator-1 (edr1l-EST). Transfection of HEK293 cells with a Wld(S)-eGFP construct produced similar changes in mRNA levels for these and seven other genes, suggesting that regulation of gene expression by Wld(S) is conserved across different species, including humans. Similar modifications in mRNA levels were mimicked for some of the genes (including pttg1) by 1 mm nicotinamide adenine dinucleotide (NAD). However, expression levels of most other genes (including edr1l-EST) were insensitive to NAD. Pttg1(-/-) mutant mice showed no neuroprotective phenotype. Transfection of HEK293 cells with constructs comprising either full-length Nmnat-1 or the truncated Ube4b fragment (N70-Ube4b) demonstrated selective effects of Nmnat-1 (down-regulated pttg1) and N70-Ube4b (up-regulated edr1l-EST) on mRNA levels. Similar changes in pttg1 and edr1l-EST were observed in the mouse NSC34 motor neuron-like cell line following stable transfection with Wld(S). Together, the data suggest that the Wld(S) protein co-regulates expression of a consistent subset of genes in both mouse neurons and human cells. Targeting Wld(S)-induced gene expression may lead to novel therapies for neurodegeneration induced by trauma or by disease in humans.
Human Molecular Genetics 03/2006; 15(4):625-35. · 7.64 Impact Factor
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ABSTRACT: We have used site-directed mutagenesis of amino acids located within the S1 and S2 ligand binding domains of the NR2A N-methyl-D-aspartate (NMDA) receptor subunit to explore the nature of ligand binding. Wild-type or mutated NR1/NR2A NMDA receptors were expressed in Xenopus laevis oocytes and studied using two electrode voltage clamp. We investigated the effects of mutations in the S1 and S2 regions on the potencies of the agonists L-glutamate, L-aspartate, (R,S)-tetrazol-5yl-glycine, and NMDA. Mutation of each of the corresponding residues found in the NR2A receptor subunit, suggested to be contact residues in the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit, caused a rightward shift in the concentration-response curve for each agonist examined. None of the mutations examined altered the efficacy of glutamate as assessed by methanethiosulfonate ethylammonium potentiation of agonist-evoked currents. In addition, none of the mutations altered the potency of glycine. Homology modeling and molecular dynamics were used to evaluate molecular details of ligand binding of both wild-type and mutant receptors, as well as to explore potential explanations for agonist selectivity between glutamate receptor subtypes. The modeling studies support our interpretation of the mutagenesis data and indicate a similar binding strategy for L-glutamate and NMDA when they occupy the binding site in NMDA receptors, as has been proposed for glutamate binding to the GluR2 AMPA receptor subunit. Furthermore, we offer an explanation as to why "charge conserving" mutations of two residues in the binding pocket result in nonfunctional receptor channels and suggest a contributing molecular determinant for why NMDA is not an agonist at AMPA receptors.
Molecular Pharmacology 06/2005; 67(5):1470-84. · 4.88 Impact Factor
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ABSTRACT: NR1/NR2D NMDA receptors display unusually slow deactivation kinetics which may be critical for their role as extrasynaptic receptors. A threonine to alanine point mutation has been inserted at amino acid position 692 of the NR2D subunit (T692A). Recombinant NR1a/NR2D(T692A) NMDA receptors have been expressed in Xenopus laevis oocytes and their pharmacological and single-channel properties examined using two-electrode voltage-clamp and patch-clamp recording techniques. Glutamate dose-response curves from NR1a/NR2D(T692A) receptor channels produced an approximately 1600-fold reduction in glutamate potency compared to wild-type NR1a/NR2D receptors. There was no change in Hill slopes or gross reduction in mean maximal currents recorded in oocytes expressing either wild-type or mutant receptors. The mutation did not affect the potency of the co-agonist glycine. The shifts in potency produced by NR2D(T692A) containing receptors when activated by other glutamate-site agonists such as aspartate or NMDA were 30- to 60-fold compared to wild-type. Single-channel conductance levels of NR1a/NR2D(T692A) mutant receptors were indistinguishable from wild-type NR2D-containing channels. Additionally NR1a/NR2D(T692A) receptors showed the transitional asymmetry that is characteristic of NR2D-containing NMDA receptors. Rapid applications of glutamate on outside-out patches containing NR1a/NR2D(T692A) receptors produced macroscopic current deactivations that were about 60-fold faster than wild-type NR1a/NR2D receptors. Our results suggest that this conserved threonine residue plays a crucial role in ligand binding to NMDA NR2 receptor subunits and supports the idea that the slow decay kinetics associated with NR1a/NR2D NMDA receptors can be explained by the slow dissociation of glutamate from this NMDA receptor subtype.
The Journal of Physiology 08/2004; 558(Pt 1):45-58. · 4.72 Impact Factor
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ABSTRACT: Ionotropic glutamate receptors (iGluRs) mediate the vast majority of fast excitatory synaptic transmissions within the mammalian central nervous system (CNS). As for other ion channel protein families, there has been astounding progress in recent years in elucidating the details of protein structure through the crystallization of at least part of the ion channel protein complex. The result is a new framework for the interpretation of both classic and emerging functional data. Here we summarize, compare, and contrast recent findings for the AMPA, kainate, and NMDA subtypes of glutamate receptor ion channels, with an emphasis on the functional and structural aspects of how agonist binding controls channel gating.
Critical Reviews in Neurobiology 02/2004; 16(3):187-224.
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York Rudhard,
Matthias Kneussel,
Mohammed A Nassar,
Georg F Rast,
Alexander J Annala, Philip E Chen,
Cezar M Tigaret,
Isabel Dean,
Juergen Roes,
Alasdair J Gibb,
Stephen P Hunt,
Ralf Schoepfer
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ABSTRACT: Precise refinement of synaptic connectivity is the result of activity-dependent mechanisms in which coincidence-dependent calcium signaling by NMDA receptors (NMDARs) under control of the voltage-dependent Mg2+ block might play a special role. In the developing rodent trigeminal system, the pattern of synaptic connections between whisker-specific inputs and their target cells in the brainstem is refined to form functionally and morphologically distinct units (barrelettes). To test the role of NMDA receptor signaling in this process, we introduced the N598R mutation into the native NR1 gene. This leads to the expression of functional NMDARs that are Mg2+ insensitive and Ca2+ impermeable. Newborn mice expressing exclusively NR1 N598R-containing NMDARs do not show any whisker-related patterning in the brainstem, whereas the topographic projection of trigeminal afferents and gross brain morphology appear normal. Furthermore, the NR1 N598R mutation does not affect expression levels of NMDAR subunits and other important neurotransmitter receptors. Our results show that coincidence detection by, and/or Ca2+ permeability of, NMDARs is necessary for the development of somatotopic maps in the brainstem and suggest that highly specific signaling underlies synaptic refinement.
Journal of Neuroscience 04/2003; 23(6):2323-32. · 7.11 Impact Factor
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ABSTRACT: Alpha-synuclein belongs to a family of structurally related proteins expressed highly in the brain and is the major component of filamentous deposits present in a range of neurodegenerative diseases (synucleinopathies). It has been implicated in learning and memory, yet the physiological role of this protein is still unclear. It was recently found that a subpopulation of C57BL/6J mice carries a chromosomal deletion of the alpha-synuclein locus, often unknown to the experimenter. As genetically engineered mice are often backcrossed with C57BL/6J animals for learning and memory experiments, we studied the importance of alpha-synuclein in spatial learning tasks by examining the performance of alpha-synuclein-/- mice in the hidden platform reference memory version of the watermaze. Our data show that alpha-synuclein-/- mice had no significant impairment in performance during training or probe trials, compared with wild-type littermates. Therefore, we conclude that alpha-synuclein is not essential for this type of spatial learning.
European Journal of Neuroscience 08/2002; 16(1):154-8. · 3.63 Impact Factor
