Fig 2: GluN1 hinge mutants disrupt the EphB2–GluN1 interaction in neurons.a Representative images of dendrites of DIV6-9 cortical neurons transfected with EGFP–GluN1 (WT, Quintuple, or Sextuple), mCherry, GluN2B, and CRISPR constructs to knock out endogenous GluN1. The control condition is WT GluN1-transfected treated with control reagents instead of ephrin-B2. The other three conditions were treated with ephrin-B2 for 45 min to induce the interaction between the NMDAR and EphBs. Top row panels show the colocalization between EGFP–GluN1 and EphB2 in white. Second row panels show the high contrasted images of EGFP–GluN1 puncta in green. Third row panels show the high contrasted images of endogenous EphB2 in magenta. Last row panels show mCherry. Outlines of morphology are shown in cyan. Scale bar = 5 µm. b Quantification of EphB2 density (puncta number per 100 µm) in indicated groups (***p < 0.005, ****p < 0.0001, ANOVA followed by Tukey’s; green dots represent Control n = 14; WT n = 21 cells; Quintuple n = 28; Sextuple n = 18). c Quantification of EGFP–GluN1 density (puncta number per 100 µm) in indicated groups (p = 0.5958, ANOVA; green dots represent Control n = 14; WT n = 21 cells; Quintuple n = 28; Sextuple n = 18). d Quantification of the effects of the different GluN1 mutants on colocalization (puncta number per 100 µm) between GluN1 and EphB2 (***p < 0.005, ****p < 0.0001, ANOVA; green dots represent Control n = 14; WT n = 21 cells; Quintuple n = 28; Sextuple n = 18).

Fig 3: Specific hinge region amino acid residues are responsible for the EphB2–GluN1 interaction in HEK293T cells.a Surface charge maps of GluN1 NTD hinge region of the indicated GluN1 single point mutants in order of increasing negative charged in the hinge region (left (positive) to right (negative)), with blue representing positive charge and red representing negative charge. Yellow outline indicates the location of the six key hinge region amino acids in WT GluN1. b Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated Myc-GluN1 single point mutants, together with GluN2B, FLAG-EphB2, and EGFP. The upper panels show PLA signal alone. The lower panels are merged images of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. c Quantification of the effects of GluN1 mutants on PLA puncta number. PLA puncta number are quantified by counting the number of puncta per 100 µm2 in EGFP+ cells and normalizing to the WT condition (****p < 0.0001, ANOVA; green dots represent n = 30 cells for each condition). d Surface charge maps of GluN1 NTD hinge region of the indicated GluN1 double point mutants as in a (left (positive) to right (negative)). Yellow outline indicates the location of the six key hinge region amino acids in WT GluN1. e Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated Myc-GluN1 double point mutants, together with GluN2B, FLAG-EphB2, and EGFP. The upper panels show PLA signal alone. The lower panels are merged images of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. f Quantification of the effects of GluN1 mutants on PLA puncta number. PLA puncta number are quantified by counting the number of puncta per 100 µm2 in EGFP+ cells and normalizing to the WT condition (****p < 0.0001, ANOVA; green dots represent n = 30 cells for each condition).

Fig 4: GluN1 charge mutants show disrupted EphB–NMDAR interaction in neurons.a Representative images of dendrites of DIV6-9 cortical neurons transfected with EGFP–GluN1 (WT, I272A, or N273A/R337D), mCherry, GluN2B, and CRISPR constructs to knock out endogenous GluN1. The control condition is WT GluN1-transfected treated with control reagents instead of ephrin-B2. The other three conditions were treated with ephrin-B2 for 45 min to induce the interaction between the NMDAR and EphBs. Top row panels show the colocalization between EGFP–GluN1 and EphB2 in white (arrows indicate examples of colocalized puncta). Second row panels show the high contrasted images of EGFP–GluN1 in green. Third row panels show endogenous EphB2 in magenta. Last row panels show mCherry. Outlines of morphology in cyan. Scale bar = 5 µm. b Quantification of EphB2 density (puncta number per 100 µm) in indicated groups (*p < 0.05, ANOVA; green dots represent n = 30 cells for each condition). c Quantification of EGFP–GluN1 density (puncta number per 100 µm) in indicated groups (p = 0.0128, ANOVA followed by Tukey’s; comparisons showed no significant differences; green dots represent n = 30 cells for each condition). d Quantification of the effects of the different GluN1 mutants on colocalization (puncta number per 100 µm) between GluN1 and EphB2 (*p < 0.05, ANOVA; green dots represent n = 30 cells for each condition).

Fig 5: pH-sensitive histidine mutants in the hinge region elucidate a charge-dependent mechanism.a Representative images of PLA results in HEK293T cells. HEK293T cells were transfected with the indicated Myc-GluN1 single point mutants, together with GluN2B, FLAG-EphB2, and EGFP. Cells were treated with media at either pH 5.0 or pH 7.3 for 30 min before PLA. Upper panels: PLA signal alone. Lower panels: merge of EGFP in green and PLA signal in magenta. Scale bar = 10 µm. b Quantification of the effects of GluN1 mutants and pH on PLA puncta number (****p < 0.0005, ANOVA; green dots represent n = 30 cells for each condition). c Surface representation models (top) and charge maps (bottom) of WT GluN1 and R337H GluN1 predicted at neutral pH. d Model of experimental design. The same neurons were imaged for both pH conditions. e Representative FRAP images at different time points of DIV21–23 cortical neurons transfected with EGFP–GluN1 (WT or R337H) together with GluN2B, CRISPR construct targeting endogenous GluN1, and mCherry. FRAP of GluN1 puncta was conducted in the same neurons at pH 5.0 (H-positive) and pH 7.3 (H-neutral) and the order of pH presentation varied. Recovery of bleached spine puncta (magenta circle) was monitored for 15 min at 10s intervals. Scale bar = 2 µm. f Left: Quantification of the recovery curve of GluN1 WT or R337H mutant in ACSF at pH 7.3 and pH 5.0 in DIV21–23 cortical neurons. Graphs represent mean intensity and show fit (****p < 0.0001, Kolmogorov–Smirnov (KS) nonparametric test). Right: Quantification of the mobile fraction of EGFP–GluN1 spine puncta at 15 min after photobleaching (*p = 0.0398, WT-pH7.3 vs. R337H-pH7.3; ANOVA; green dots represent WT-pH5.0 n = 26 puncta; R337H-pH5.0 n = 24; WT-pH7.3 n = 23; R337H-pH7.3 n = 35). Error bars show S.E.M. g Left: Quantification of the recovery curve of GluN1 WT or I272H mutant in ACSF at pH7.3 and pH5.0 in DIV21–23 cortical neurons. Graphs represent mean intensity and show fit. (p = 0.8148, WT-pH7.3 vs. I272H-pH7.3, Kolmogorov–Smirnov (KS) nonparametric test). Right: Quantification of the mobile fraction of EGFP–GluN1 spine puncta at 15 min of FRAP (p = 0.9839; WT-pH7.3 vs. I272H-pH7.3; ANOVA; green dots represent WT-pH5.0 n = 26 puncta; I272H-pH5.0 n = 22; WT-pH7.3 n = 23; I272H-pH7.3 n = 30). Error bars show S.E.M.