Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Characterizing the cellular localization of the NMDA receptors regulating [ 3 H] ‐NE release in young rat cortical brain slices. 1 mM Glu‐stimulated‐[ 3 H]‐NE releases in the cerebral cortex tissue slices from young rats ( n = 3–7) in the presence 1 & 3 μM of the TTX, voltage‐gated Na channel blocker, 10 μM MK‐801, and a combination of MK‐801 and TTX. Data are expressed as mean (±SEM) of net fractional release (stimulated—basal), with each data point representing a duplicate from one animal. Data were analyzed using a mixed‐effect analysis followed by Dunnett's multiple comparison test **** p < 0.0001. NE, norepinephrine; Glu, glutamate; TTX, tetrodotoxin.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Comparison

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: The stimulatory effect of Glutamate Vs. NMDA on [ 3 H] ‐NE release in young rat cortical brain slices. In (a), the concentrations‐response curves of glutamate and NMDA‐stimulated [ 3 H] ‐NE releases in the cerebral cortex tissue slices from young rats ( n = 4). In (b), the 1 mM glutamate and NMDA stimulated NE release in the presence and absence of 1.2 mM magnesium in the cerebral cortex tissue slices from young rats ( n = 3). Data were analyzed using an unpaired t ‐test. * p ≤ 0.05, ** p ≤ 0.01 NE, norepinephrine; Glu, glutamate; NMDA, N‐methyl‐ d ‐aspartate; Mg 2+ , magnesium.

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques:

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Age‐associated changes in the expression of NMDA receptors freely solubilize subunits in the cortical rat tissue homogenate. A representative Western blot for NMDA receptors subunits in the cerebral cortex in (a); and in (b) quantified results for GluN1, GluN2A, and GluN2B expressions in young and aged rats ( n = 5). Data are expressed as mean (±SEM) normalized to young rats, with each data point representing a duplicate from one animal. Data were analyzed using an unpaired t ‐test. * p ≤ 0.05. NMDA, N‐methyl‐ d ‐aspartate).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Expressing, Western Blot

Journal: Journal of Neurochemistry

Article Title: Pharmacological target sites for restoration of age‐associated deficits in NMDA receptor‐mediated norepinephrine release in brain

doi: 10.1111/jnc.16280

Figure Lengend Snippet: Effect of aging on [ 3 H]‐MK‐801 binding to NMDA receptors in the young (2–3 months old) and aged (18–24 months old) rat cortical tissue membrane. In (a) saturation curve of [ 3 H]‐MK‐801 binding to NMDA receptors in young rats in the presence of 10 μM Glu and Gly ( n = 4). The non‐linear least‐squares fitting of the saturation isotherm yielded K d and B max values of 1.8 nM and 970 fmol/mg of protein, respectively. Both total and non‐specific binding of [ 3 H]‐MK‐801 is shown in the curve, and the specific [ 3 H]‐MK‐801 binding is presented with 95% CI in dotted lines. Inset: Saturation data graphed as Scatchard plots. Whereas in (b), the binding of 10 nM [ 3 H]‐MK‐801 in +/− 10 μM Glu and Gly in young and aged rats ( n = 7), each performed in triplicate and repeated twice. In (c), the % increases after subtracting baseline binding from the binding in the presence of 10 μM Glu and Gly. Baseline Binding values represent [ 3 H]‐MK‐801 binding without exogenous addition of Glu and Gly. Data were analyzed using a Mixed‐effect analysis followed by Tukey's multiple comparison tests. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001; whereas the % of increase over the baseline was analyzed using an unpaired t ‐test: ** p ≤ 0.01. NMDA, N‐methyl‐d‐aspartate; Glu, Glutamate; Gly, Glycine; K d , dissociation constant; B max , Maximum Binding; n H , Hill Coefficient).

Article Snippet: The NMDA receptor NR2A and NR2B subunit polyclonal antibodies were used alongside the NR1 monoclonal antibody (PhosphoSolutions, CO, USA; Cat # 1805‐NR1).

Techniques: Binding Assay, Membrane, Comparison

Journal: Antioxidants (Basel, Switzerland)

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: Figure 2. (A) The chemical structure of ergotamine. (B) The H2O-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 (Basel, Switzerland)

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: Figure 3. 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 (Basel, Switzerland)

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: Figure 4. 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 ergo- tamine 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, Residue

Journal: Antioxidants (Basel, Switzerland)

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: Figure 5. 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: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , The representative immunoblots ( a ) and quantitative analyses ( b ) of Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 4 per group. c , RT–qPCR assays mRNA expression of the Glun1, Glun2A, Glun2B and Syn1 in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. d , ChIP–qPCR analysis of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B, Glun2C and Syn1 promoters in the hippocampus of the WT, 3xTg-AD and 3xTg-AD+β-OHB mice, n = 5 per group. e , f , Supplementing with β-OHB could increase the density of 3xTg-AD dendritic spines detected by Golgi-cox staining; the representative images ( e ) and quantitative analysis ( f ) of spine, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , The Sholl analysis showed the synaptic complexity of neurons after supplementing with β-OHB in 3xTg-AD mice; the representative images ( g and i ) and the quantitative analysis ( h and j ), n = 5 per group, two fields per mice. Scale bar, 50 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and f . Two-way ANOVA followed by Bonferroni’s post hoc test for i and k . * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Western Blot, Quantitative RT-PCR, Expressing, ChIP-qPCR, Staining

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a – c , The HMGCS2 upregulation promotes the protein ( a and b ) and mRNA ( c ) expression of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 4 or 5 per group. d , e , ChIP–qPCR analyses of the enrichment of H3K9bhb at Glun1, Glun2A, Glun2B and Syn1 promoters in the primary neurons of the WT, 3xTg-AD and 3xTg-AD + HMGCS2 mice n = 5 per group ( d ) and representative gel images from ChIP–qPCR assays ( e ). f – j , The HMGCS2 upregulation promotes the expression of Syn1 (scale bar, 25 μm) ( f ); n = 10 cells per group in MAP2 immunofluorescence ( g ) and quantitative analysis ( h ), n = 10 cells per group and SYP ( i and j ), n = 10 cells per group, scale bar, 15 μm. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d and j . Two-way ANOVA followed by Bonferroni’s post hoc test for h . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Expressing, ChIP-qPCR, Immunofluorescence

Journal: Experimental & Molecular Medicine

Article Title: HMGCS2-dependent β-OHB/H3K9bhb ameliorates synaptic plasticity and cognition in Alzheimer’s disease

doi: 10.1038/s12276-026-01664-9

Figure Lengend Snippet: a , b , A western blot analysis ( a ) of hippocampal lysates shows that HMGCS2 upregulation increases the protein levels ( b ) of H3K9bhb, Glun1, Glun2A, Glun2B, Syn1 and PSD95, n = 3 per group. c , The ChIP–qPCR analysis of H3K9bhb enrichment at the promoters of Glun2A , Glun2B , Syn1 and PSD95 in the four groups, n = 5 per group. d , The mRNA levels of Glun1, Glun2A, Glun2B, Syn1 and PSD95 in the hippocampus, as determined by RT–qPCR, n = 5 per group. e , f , Golgi staining reveals increased dendritic spine density in 3xTg-AD mice following overexpression of HMGCS2; representative images ( e ) and quantification ( f ) are shown, n = 5 per group, three fields per mice. Scale bar, 5 μm. g – j , A behavioral assessment of spatial learning and memory using the MWM, NOR and contextual fear conditioning tests: area under the curve (AUC) of escape latency during MWM training of day 1–6 ( g ), escape latency on day 7 of the MWM test ( h ), NOR discrimination index ( i ), freezing time on day 7 in the contextual fear conditioning test ( j ), n = 8 per group. Data are shown as mean ± s.e.m. One-way ANOVA followed by Bonferroni’s post hoc test for b – d , f and g – j . * P < 0.05, ** P < 0.01 , *** P < 0.001, **** P < 0.0001; ns, not significant.

Article Snippet: GluN2A , NMDAR2A , Poly- , 1:1000 , Proteintech , 28525-1-AP.

Techniques: Western Blot, ChIP-qPCR, Quantitative RT-PCR, Staining, Over Expression

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: Figure 1. 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, Membrane, 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: Figure 2. 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 × 104 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: Figure 3. 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 2 for n to create averaged data per construct, 3 × 104 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: Figure 4. 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 × 104 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: Figure 5. 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 EC50 (Pearson’s coefficient of determination r2 = 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: Membrane, Western Blot, Control, 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: Figure 6. 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 × 104 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. 3c) and the response of the mutant before (solid line) and after (dashed line) the addition of PAM. p < 0.0001 for LogEC50 for each construct + PAM when compared to without PAM addition. Data averaged from n = 12 wells for each construct, 3 × 104 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, Comparison, Construct, Concentration Assay