Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: ( A ) The chemical structure of ergotamine. ( B ) The H 2 O-injected oocytes did not cause any change with the treatment of 100 µM glutamate ( n = 6–8 oocytes from four different frogs). ( C – F ) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors ( C ) NR1a/NR2A, ( D ) NR1a/NR2B, ( E ) NR1a/NR2C, and ( F ) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner ( n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Recombinant

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: The value of I max , IC 50 , and n H (Hill coefficient) of ergotamine for the glutamate-evoked current in each recombinant receptor. Values represent means ± S.E.M. ( n = 6–8/group). IC 50 , Hill’s coefficient; I max value as determined as described in Materials and methods.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Recombinant

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: The predicted docking sites and binding energy of NR1a/NR2A receptor and ergotamine.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Computational molecular modeling of ergotamine docked to the human NR1a/NR2A receptor. ( A , C ) Side views of the docked ergotamine complex with NMDA channel. ( B , D ) Binding pocket and docking results of ergotamine and NMDA channel, respectively.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Binding Assay

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Predicted binding mode of ergotamine and all the favorable interactions with several residues in the active site of the human NR1a/NR2A receptor. ( A , B ) Interaction between ergotamine and wild-type NR1a/NR2A. ( C ) Residues in wild-type NR1a/NR2A receptor interacting with the ergotamine molecule. ( D ) Change in the interaction distance of ergotamine in mutant-type NR1a/NR2A receptor. Based on the change in this distance, the residue that directly interacts with ergotamine was identified.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Binding Assay, Mutagenesis

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Effect of ergotamine on the glutamate-evoked current of wild-type NR1a/NR2A receptor and its various mutants.

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques:

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: The inward current of several mutant types on the glutamate-evoked current of NR1a/NR2A receptor with or without ergotamine. Representative traces of current induced by application of glutamate (100 µM) alone or together with ergotamine (100 µM) for various mutants: ( A ) mutant 1 (NR1a subunit W167A and NR2A wild-type), ( B ) mutant 2 (NR1a subunit H168A and NR2A wild-type), ( C ) mutant 3 (NR1a subunit V169A and NR2A wild-type), ( D ) mutant 4 (NR1a wild-type and NR2A subunit P435A), ( E ) mutant 5 (NR1a wild-type and NR2A subunit N466A), and ( F ) mutant 6 (double mutant-type NR1a subunit V169A and NR2A subunit N466A). Experiments were performed separately, and data were collected from several oocytes ( n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Mutagenesis

Journal: Antioxidants

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors

doi: 10.3390/antiox11081471

Figure Lengend Snippet: ( A ) Concentration-response curves for the effect of ergotamine on expressed NMDA receptors. The percentage inhibition by ergotamine was calculated based on the average of peak inward current elicited by glutamate and that of peak inward current elicited by glutamate and ergotamine. Each point represents the mean ± S.E.M. ( n = 6–8 oocytes from four different frogs). ( B ) Concentration–response curves for the inhibition of ergotamine on glutamate-induced inward current of NR1a/NR2A mutant types. NR1a (W167A, H168A, and V169A) and NR2A mutants (P435A and N466A) were cross-combined between wild-type/mutants of each subunit. Ergotamine reduced I Glu in a concentration-dependent manner in the wild-type. Experiments were performed at a holding potential of −80 mV. The value of IC 50 , I max , and n H (Hill coefficient) are shown in . Each point represents the mean ± S.E.M. ( n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Concentration Assay, Inhibition, Mutagenesis

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: The values are IC 50 s for inhibition of Ca 2+ responses mediated by GluN2A receptors expressed in HEK cells.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Cells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. Curves for inhibition of the Ca 2+ response in GluN2A-expressing cells were derived from fits to the Hill equation using GraphPad Prism (v6.00 for Mac, GraphPad Software, La Jolla California USA). Whereas MPX-004 and MPX-007 achieve full inhibition of the GluN2A Ca 2+ response by ~ 3 μM, TCN-201 never inhibits more than ~40% of the response. Each data point is a mean (± standard deviation) of data from 20–86 experiments).

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Expressing, Derivative Assay, Software, Standard Deviation

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: C ells were stimulated with glutamate and glycine (3 μM each) in the presence of compounds at a range of concentrations. IC 50 for inhibition of the Ca 2+ response in GluN2A-expressing cells was fitted to the Hill equation using CDD Vault. For GluN2B or GluN2D no IC 50 could be determined, so the effect of each compound at 10 μM is shown as the % inhibition of the Ca 2+ response (note that negative % inhibition represents an increase in Ca 2+ response over glutamate plus glycine alone). Values are the mean IC 50 or % ± SEM with the number of replicate curves indicated in parentheses. ND- not determined.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Expressing

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Oocytes were exposed to MPX-004 or MPX-007 at concentrations from 10 nM to 10 μM as indicated. Inhibition curves were generated using GraphPad Prism and IC 50 values were generated using CCD Vault. The IC 50 s for inhibition of GluN2A-mediated currents were 198 ± 17 and 143 ± 10 nM for MPX-004 and MPX-007, respectively. Each data point is a mean (± standard deviation) of data from 4–12 oocytes.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques: Inhibition, Generated, Standard Deviation

Journal: PLoS ONE

Article Title: MPX-004 and MPX-007: New Pharmacological Tools to Study the Physiology of NMDA Receptors Containing the GluN2A Subunit

doi: 10.1371/journal.pone.0148129

Figure Lengend Snippet: Effect of MPX-004 on the ratio of NMDA receptor- to AMPA receptor-mediated synaptic currents (NMDAR/AMPAR ratio) at layer 4-to-2/3 synapses in mouse visual cortex.

Article Snippet: The human GRIN1 gene transcript variant NR1-3 (Origene, SKU#: SC115601, Rockville, MD), GRIN2A gene (Origene, SKU#: SC326381), GRIN2B gene (B’SYS, Witterswil, Switzerland), GRIN2C gene (NCBI locus NM_000835, optimized for oocyte expression and synthesized by GenScript, Piscataway, NJ) and GRIN2D gene (NCBI locus NM_000836, sequence optimized and synthesized by GenScript) were linearized and transcribed using the mMessage mMachine kit (Life Technologies) and promoters SP6 (for GluN1) and T7 (for GluN2A-2D). cRNAs were mixed together at a ratio of 1 GluN1:2 GluN2.

Techniques:

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: CADD scores and protein modelling predict stronger functional effects for EAS-associated GRIN2A mutations than in controls. ( a ) Protein structure model of NMDAR (PDB ID 4TLL): GluN1 (grey and green), GluN2 (B in this structure) (blue and red). Membrane would be horizontal in this image with the NTD and ABD in the extracellular space. Intracellular C-Terminal domain would be below the transmembrane domain (not present in this structure). ( b ) Schematic linear representation of GluN2A with the domains annotated. Black rectangles indicate transmembrane domains. Plot of scaled CADD scores against GluN2A amino acid position for missense variants. Black dots represent scores for 65/6474 individuals from the Exome Variant Server (EVS) that had missense variants in GRIN2A and coloured symbols are scores for variants found in individuals with EAS disorders. The horizontal dotted line indicates the scaled CADD score cut off of 20 for a highly likely deleterious variant. ( c ) Protein structure model of the NTD of NMDAR (PDB ID 3QEL): GluN1 (grey), GluN2 (B in this structure) (red). Mutations considered in this domain highlighted (conserved between GluN2A and B). ( d ) Protein structure model of the LBD of NMDAR (PDB ID 2A5T): GluN1 (grey), GluN2A (blue). Mutations considered in this paper highlighted, as well as agonists.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Functional Assay, Variant Assay

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Selected GRIN2A mutations protect against glutamate-induced toxicity in HEK cells. ( a ) Superimposed bright field and pseudocolour red images, indicating fluorescent Cytotox Red dye in dead cells, of HEK cells transiently transfected with various GRIN2A mutant constructs, or empty vector. Free glutamate and glycine in the culture media causes cell death in those expressing functional NMDARs over 48 hours. Images captures at 20x using the IncuCyte live-cell imaging system. ( b ) Representative time course of cell death, normalised to initial confluency in each well, n = 5 wells, Mean ± SEM. ( c ) Plot of cell mortality for GRIN2A mutants normalised to initial confluency per well. Mutants P79R, C231Y, C436R, G483R, M705V, D731N and I814T are protective against glutamate toxicity either due to reduced trafficking and/or reduced functionality of the receptors. ***p < 0.001 Dunnett’s corrected one-way ANOVA. Averaged data from n = 15 wells per construct except for G483R, E714K, D933N and N976S where n = 12 wells, 3 × 10 4 cells/well. Mean ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Transfection, Mutagenesis, Construct, Plasmid Preparation, Expressing, Functional Assay, Live Cell Imaging

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: GRIN2A mutations alter the response to glutamate and glycine. ( a,b ) Pseudocolour images and representative single-cell traces from calcium-flux imaging for HEK cells transfected with either WT or G483R GRIN2A showing different calcium responses caused by the application of increasing concentrations of glutamate (30 nM–30 µM red arrows). Individual cell traces in cyan and the mean response in red. ( c , d ) Normalised concentration response curves (CRCs) to increasing concentrations of glutamate from single cell calcium-flux imaging. In ( c ) 4 mutants; P79R, C231Y, C483R and M705V have reduced agonist potency, while C436R and D731N show no response to glutamate. Four mutant constructs ( d ), show an unaltered response to glutamate. ( e,f ) Normalised CRCs to increasing glycine concentration with constant 3 µM glutamate – the mutations show similar responses as to glutamate. See Table for n to create averaged data per construct, 3 × 10 4 cells/well. Error bars ± SEM. ( g ) Representative continuous voltage-clamp recordings obtained from HEK cells transfected with WT or G483R GRIN2A plasmid. Bars above the recording indicate glutamate application (co-applied with 30 µM glycine). Application of increasing concentrations of glutamate shows a progressive increase in current observed, the sensitivity of which is shifted to higher concentrations of glutamate in the G483R mutant compared to WT. ( h ) Normalised CRC to glutamate as recorded in ( g ) indicates the same response as for single cell calcium imaging (n = 3).

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Imaging, Transfection, Concentration Assay, Mutagenesis, Construct, Plasmid Preparation

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: GRIN2A mutations alter the number of cells responding to glutamate. ( a–d ) Each panel contains pseudocolour images of HEK cells transfected with WT or mutant GRIN2A showing co-localisation of responses to application of 100 µM glutamate +30 µM glycine (green - activation of surface-expressing GluN2A receptors) and subsequently 100 µM MgATP (red - activation of endogenous P2Y receptors). Panels also show single-cell traces from the same experiment, indicating the different effects of these agonists on cells transfected with ( a ) WT, ( b ) M705V, ( c ) C231Y or ( d ) C436R mutants. Individual cell traces displayed in cyan and the mean response shown in red. ( e ) Ratio of the number of transfected HEK cells responding to 100 µM glutamate +30 µM glycine subsequent to 100 µM MgATP. Dunnett’s corrected one-way ANOVA compared to WT, **p < 0.01, ***p < 0.001, ns = non-significant. Data averaged from n = 12 wells, 3 × 10 4 cells/well, for each construct over 2 assays. Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Transfection, Mutagenesis, Activation Assay, Expressing, Construct

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: GRIN2A mutations reduce total protein levels and membrane trafficking of GluN2A. ( a ) Representative Western blot of HEK lysates probed with anti-GluN2A antibody (top), and anti-GAPDH (bottom) as a loading control. Bands around 180 kDa indicate GluN2A. WT, wild type, UT – untransfected. Right is Amersham Full-Range rainbow molecular weight marker with the blot imaged in visible light. ( b ) Plot of amount of GluN2A protein, normalised to WT, from Western blotting of total cell lysates of transiently co-transfected HEK cells 48-hours post transfection. Average of 3 blots from 3 independent transfections. Error bars indicated SEM. ( c ) Fixed and immunolabelled co-transfected HEK cells with anti-HA antibody (red) to detect surface GluN2A expression, and fixed, permeabilised and immunolabelled with the same antibody to detect total GluN2A protein levels. Scale bar 25 µm. Nuclei stained with Hoechst (blue). ( d ) Quantitation of surface and total GluN2A protein levels, averaged over the total number of cells analyzed for each condition (n is between 903 to 2255 cells), reveals greatly reduced surface expression of GluN2A as measured by fluorescence intensity. Dunnett’s corrected one-way ANOVA for membrane or total intensity as compared to WT *p < 0.05, **p < 0.01, ***p < 0.001, ns = non-significant. Average ± SEM. ( e ) Relative surface levels of NMDARs correlated with the log of glutamate EC 50 (Pearson’s coefficient of determination r 2 = 0.77, two-tailed p = 0.002). ( f ) Normalised CRC from single-cell calcium-flux imaging. Response to increasing concentrations of glutamate from HEK cells co-transfected with decreasing quantities of WT GRIN2A per well (100% = 320 ng) with standard 320 ng GRIN1 per well. Response is compared to mutant P79R. Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Western Blot, Molecular Weight, Marker, Transfection, Expressing, Staining, Quantitation Assay, Fluorescence, Two Tailed Test, Imaging, Mutagenesis

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Pharmacological rescue of functional deficits can be achieved for selected GRIN2A mutants. Examples of single-cell imaging of HEK transfected with (a) WT and (b) C231Y mutant showing calcium-influx response to 300 nM glutamate before and after incubation with 1 µM PAM and in comparison to maximal 30 µM glutamate. (c) Graph of single cell imaging data showing response of WT and mutants P79R, C231Y and G483R to 300 nM glutamate with and without 1 µM PAM. Bonferroni corrected ANOVA ***p < 0.001, n = 15 wells (over 3 assays) for each construct, 3 × 10 4 cells/well. Mean ± SEM. (d) Examples of single cell imaging of HEK transfected with C231Y mutant showing calcium-influx response to increasing concentrations of glutamate (30 nM, 100 nM, 300 nM, 1 µM, 10 µM and 30 µM arrows) top, and below, increasing concentrations of glutamate (30 nM, 100 nM, 300 nM, 1 µM and 30 µM arrows) with constant 1 µM PAM. Individual cell traces displayed in cyan and the mean response shown in red. (e–h) Concentration response curves of mutant (P79R, C231Y, G483R or M705V) GRIN2A transfected in HEK cells to increasing concentrations of glutamate (30 nM to 30 µM) during incubation with 1 µM PAM recorded from single-cell calcium-flux imaging. Each graph shows the response of the WT GRIN2A construct with no PAM in red (curve from Fig. ) and the response of the mutant before (solid line) and after (dashed line) the addition of PAM. p < 0.0001 for LogEC 50 for each construct + PAM when compared to without PAM addition. Data averaged from n = 12 wells for each construct, 3 × 10 4 cells/well, (over 3 assays). Error bars ± SEM.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques: Functional Assay, Imaging, Transfection, Mutagenesis, Incubation, Construct, Concentration Assay

Journal: Scientific Reports

Article Title: Epilepsy-associated GRIN2A mutations reduce NMDA receptor trafficking and agonist potency – molecular profiling and functional rescue

doi: 10.1038/s41598-017-00115-w

Figure Lengend Snippet: Summary of clinical and genetic information for studied GRIN2A mutations.

Article Snippet: Human GRIN2A (GenBank accession: NM_000833.3) cDNA clone was purchased from OriGene Technologies (Cat#: SC122120).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented and reversed mechanical allodynia in rats with SNI. a The threshold force of SNI-induced mechanical allodynia on the ipsilateral hind paw was significantly decreased on day 7 and continued until day 14. Intrathecal treatment with the NR2A-selective antagonist NVP-AAM077 (4 nmol) and the NR2B-selective antagonist Ro25-6981 (20 nmol) once daily for 14 days prevented the development of mechanical allodynia on the hind paw ipsilateral to SNI on days 3, 5, 7, 10, and 14. c A single intrathecal administration of NVP-AAM077 (4 nmol) and Ro25-6981 (20 nmol) on day 14 attenuated mechanical allodynia at 30 min after the treatment, and the effect of NVP-AAM077 was maintained for 24 h. b , d NVP-AAM077 and Ro25-6981 did not change the mechanical nociceptive threshold of the contralateral hind paw in the same rat. e A single intrathecal administration of 10 nmol MK-801 on day 14 reversed SNI-induced mechanical allodynia 30 min after the treatment. NVP, NVP-AAM077; Ro25, Ro25-6981; MK, MK-801. Data are shown as the means ± SE. * P < 0.05, ** P < 0.01 versus vehicle. # P < 0.05, ## P < 0.01 versus before the single intrathecal treatment

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: NR2A and NR2B antagonists prevented exogenous leptin-induced mechanical allodynia. a Intrathecal leptin (50 μg) treatment in naïve rats, given once daily for 7 days, induced mechanical allodynia on day 7. Coadministration of leptin with 4 nmol NVP-AAM077 or 20 nmol Ro25-6981 attenuated the behavioral changes ( n = 5). b NVP-AAM077 and Ro25-6981 alone did not change the baseline nociceptive threshold ( n = 6). lep, leptin; NVP, NVP-AAM077; Ro25, Ro25-6981. Data are shown as the means ± SE. ** P < 0.01 versus day 0

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques:

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B- but not NR2A-mediated currents in dissociated lamina II neurons in naïve rats. a Treatment with the NR2A-selective antagonist NVP-AAM077 (0.4 μM) plus the NR2B-selective antagonist Ro25-6981 (1 μM) blocked NMDAR-mediated currents ( n = 8). b Exposure to leptin (100 nM) for 5 min did not change NMDAR-mediated currents after blockade with Ro25-6981 (1 μM) ( n = 10). c Exposure to leptin (100 nM) for 5 min enhanced NMDAR-mediated currents after inhibition by 0.4 μM NVP-AAM077 ( n = 9). d Histograms showing the effect of leptin on NMDAR-mediated currents after inhibition by NVP-AAM077 or Ro25-6981. Data are shown as the means ± SE. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. * P < 0.05, ** P < 0.01 vs. vehicle; # P < 0.05 vs NVP

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Inhibition

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin enhancement of NR2B, but not NR2A, expression in cultured DRG neurons. a Immunohistochemistry results showed that administration of leptin in culture medium for 72 h upregulated NR2B expression in a dose-dependent manner (2 ng/ml leptin had the maximal enhancement effect), and cotreatment with 1 μM Ro25-6981 diminished the upregulation. b Leptin at 2 ng/ml slightly enhanced NR2A expression, which was attenuated by 0.4 μM NVP-AAM077. c – f Western blot results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated NR2B expression ( c , d ) but not NR2A expression ( e , f ) in cultured DRG neurons. The NR2B upregulation was blocked by 1 μM Ro25-6981 ( c , d ). Neither 1 μM Ro25-6981 nor 0.4 μM NVP-AAM077 alone changed the baseline expression of NR2B or NR2A. lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. * P < 0.05 vs vehicle

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Cell Culture, Immunohistochemistry, Western Blot

Journal: Molecular Neurobiology

Article Title: Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

doi: 10.1007/s12035-020-02180-1

Figure Lengend Snippet: Leptin-mediated enhancement of nNOS expression was blocked by an NR2B antagonist. Immunohistochemistry ( a ) and Western blot ( b and c ) results showed that administration of leptin (2 ng/ml) to culture medium for 72 h significantly upregulated nNOS expression in cultured DRG neurons. The upregulation of nNOS expression by leptin was significantly prevented by coapplication of the NR2B antagonist Ro25-6981 (1 μM) and slightly attenuated by the NR2A antagonist NVP-AAM077 (0.4 μM). Ro25-6981 (1 μM) and NVP-AAM077 (0.4 μM) alone did not change baseline nNOS expression. Lep, leptin; NVP, NVP-AAM077; Ro, Ro25-6981. n = 3. Scale bar, 50 μm. ** P < 0.01 vs vehicle; # P < 0.05 vs leptin

Article Snippet: Membranes were blocked with 5% nonfat dried milk and incubated overnight (4 °C) with anti-NR2A antibody (BOSTER, China: 1:400, rabbit polyclonal), anti-NR2B antibody (Abcam, USA: 1:1000, rabbit polyclonal) [ ] and anti-nNOS antibody (BD Biosciences, USA: 1:1000, mouse monoclonal).

Techniques: Expressing, Immunohistochemistry, Western Blot, Cell Culture