Western blot of 10 µg of rat hippocampal lysate showing specific immunolabeling of the ~180k NR2A subunit of the NMDA receptor.
   Figure 4 GluN2A Expression Is Sufficient to Displace GluN2B from NMDARs (A-D) Total GluN2B expression was measured in extracts from the neocortex at the indicated stages for the indicated genotypes (n = 8 per genotype). (A) and (B) show quantitation and example blots, respectively, for GluN2A WT/WT versus GluN2A 2B(CTR)/2B(CTR) . (C) and (D) show quantitation and example blots, respectively, for GluN2A WT/WT vs. GluN2B DeltaCaMKII/DeltaCaMKII . (E-G) Ectopic GluN2A expression is sufficient to displace GluN2B from NMDARs. Young mouse (E and F) and rat (G) neurons at DIV7 were transfected with a control (beta-globin) or GluN2A-encoding vector and spermine (100 muM) potentiation of NMDAR currents measured 72 hr later. * p
   Figure 1 The GluN2B CaMKII Site Is Dispensable for the Developmental Switch In Vitro (A) Schematic of the GluN2B DeltaCaMKII amino acid changes. (B and C) Altered GluN2B phosphorylation in GluN2B DeltaCaMKII/DeltaCaMKII neurons. Cortical neuronal extracts were prepared and analyzed by western blot with the indicated antibodies, normalized to total GluN2B. (B) shows quantitation (mean +- SEM here and throughout), and (C) shows an example. * p
   Figure 1 Subcellular distribution of Rph3A and interaction with GluN2A and PSD-95. ( a ) Fluorescence immunocytochemistry of Rph3A (green) and PSD-95 (red) in DIV15 primary hippocampal neurons. On last panel (merge), co-localization points are shown in white. Scale bar, 10 mum. ( b ) Fluorescence immunocytochemistry of GluN2A (green) and Rph3A (red) in DIV15 primary hippocampal neurons. On last panel (merge), co-localization points are shown in white. Scale bar, 10 mum. ( c ) Fluorescence immunocytochemistry of eGFP-GluN2A (green), RFP-Rph3A (red) and endogenous PSD-95 (blue) in DIV15 primary hippocampal neurons transfected with eGFP-GluN2A and RFP-Rph3A ( DIV9 ). Scale bar, 10 mum. ( d ) Subcellular expression of GluN2A, PSD-95, Synaptophysin (Syn), Rph3A, Rab3A and Rab8 in rat hippocampus. H, homogenate; S1/2, supernatant 1/2; P1/2, pellet 1/2; SPM, synaptosomal plasma membrane; PSD1/2, postsynaptic density fraction 1/2. ( e , f ) Immunolabelling of Rph3A in dendritic spines of pyramidal cells in the CA1 stratum radiatum of the hippocampus. Electron microscopy images show that Rph3A is found lateral to the PSD (white arrowheads) and at different positions in dendritic spines (white arrowheads). b, bouton, s, spine. ( g ) Co-immunoprecipitation experiments on rat hippocampal P2 fractions using polyclonal GluN2A, monoclonal PSD-95 and irrelevant monoclonal Meox2 antibodies show that Rph3A is associated with both GluN2A and PSD-95.
   Figure 4 Effect of GluN2A/Rph3A complex on GluN2A synaptic availability in hippocampal neurons. ( a ) Fluorescence immunocytochemistry of GluN2A (green) and Shank (red) or GluN2B (green) and Shank in DIV15 neurons transfected with tGFP-shScramble or tGFP-shRph3A ( DIV9 ). ( b ) Bar graph representing the percentage of co-localization of GluN2A or GluN2B with Shank ( n =10-19). ( c - e ) Co-immunoprecipitation of GluN2A with PSD-95 and Rph3A in P2 fractions from primary hippocampal neurons ( DIV15 ) treated with TAT-2A-40 10 muM 30 min, showing a reduction of the interaction compared with animals treated with the control peptide TAT-Scr. The bar graphs show Rph3A/GluN2A ( d ) and PSD-95/GluN2A ( e ) co-immunoprecipitation expressed as % of TAT-Scr ( n =4). ( f ) Fluorescence immunocytochemistry of GluN2A (green) and Shank (red) in DIV15 neurons treated with TAT-Scr or 10 muM TAT-2A-40 for 30 min. ( g ) Bar graph representing the percentage of co-localization of GluN2A with Shank ( n =7-14). ( h ) Fluorescence immunocytochemistry of surface GluN2A (red) and total GluN2A (green) in DIV15 hippocampal neurons treated for 30 min with 10 muM TAT-Scr or TAT-2A-40. ( i ) Bar graph representation of the percentage of integrated density ratio GluN2A surface/total compared with the mean of TAT-Scr ( n =119-144). ( j ) Fluorescence immunocytochemistry of surface GluN2A (red) and PSD-95 (blue) in DIV15 hippocampal neurons treated for 30 min with TAT-Scr or TAT-2A-40 10 muM. ( k ) Bar graph
   Figure 6 Effect of PSD-95/Rph3A complex on GluN2A synaptic availability in hippocampal neurons. ( a - c ) Co-immunoprecipitation of Rph3A with GluN2A and PSD-95 from P2 fraction from forebrain of mice 2 h after injection with TAT-Rph3A-9c (3 nmol g -1 , i.p.) showing a reduction of both interactions compared with mice treated with the control peptide TAT-Rph3A(-VSSD). The bar graphs show GluN2A/Rph3A (left columns) and PSD-95/Rph3A (right columns) co-immunoprecipitation expressed as % of TAT-Rph3A(-VSSD); ** P
   Figure 9 Modulation of GluN2A-containing NMDAR expression at synapses in the developing rat hippocampus. ( a ) Western blot analysis of GluN2A, GluN2B, PSD-95, Rph3A and tubulin of TIF from treated rat pups hippocampus. ( b ) Bar graph representing the percentage of tubulin normalized integrated density of GluN2A, GluN2B, Rph3A and PSD-95 WB bands from TIF samples compared with their respective TIF purification TAT-Scr control ( n =3-5; unpaired Student s t -test). ( c ) Sample traces illustrating a decreased NMDA/AMPA in TAT-2A-40-treated animals (blue) compared with TAT-Scr-treated (red) animals at Schaffer collaterals to CA1 pyramidal cell synapses in acute hippocampal slices with 100 pA over 100 ms scale. ( d ) Bar graph summarizes the significant decrease in NMDA/AMPA in TAT-2A-40-treated animals (blue) compared with TAT-Scr-treated (red) animals ( n =9-12; Mann-Whitney test). ( e , f ) Sample traces ( e ) and summary bar graph ( f ) illustrating that the decay time of pharmacologically isolated NMDAR-EPSCs does not differ between TAT-Scr-treated (red) and TAT-2A-40-treated (blue) animals ( n =10-14) with 40 pA over 100 ms scale. ( g - i ) Sample traces ( g ) and summary graphs illustrating that the amplitude ( h ) and decay time ( i ) of the pharmacologically isolated NMDAR-EPSCs are equally modulated by application of TCN 210 in both conditions ( n =6-7; Wilcoxon test) with 50 pA over 200 ms scale. ( j ) Representative images show dendrites of P15 rat pups CA1 neu
   Figure 3 Cross-linking partners on the GluN1 ATD at the L1-L1 interface. Western blotting analysis of GluN1 cysteine knockout mutants coexpressed with GluN2A WT or GluN2A cysteine mutants. ( A ) The blot probed by GFP antibody. ( B ) The blot probed by GluN1 and GluN2A antibody. Open arrowhead indicates the cross-linked dimer bands. Filled arrowhead indicates positions of GluN1 or GluN2A monomer bands.

N-methyl-D-aspartate (NMDA) receptors are a class of ionotropic glutamate-gated ion channels. These receptors have been shown to be involved in long-term potentiation, an activity-dependent increase in the efficiency of synaptic transmission thought to underlie certain kinds of memory and learning. NMDA receptor channels are heteromers composed of the key receptor subunit NMDAR1 (GRIN1) and 1 or more of the 4 NMDAR2 subunits: NMDAR2A (GRIN2A), NMDAR2B (GRIN2B), NMDAR2C (GRIN2C) and NMDAR2D (GRIN2D). Alternatively spliced transcript variants encoding different isoforms have been found for this gene.