Figure 6: ND2-TM-6-8 is sufficient to disrupt the ND2–NMDAR complex.

(a) Histogram depicting normalized Venus intensity signal observed in GluN1-positive HEK293 cells transfected with GluN1ΔCTD-Vc and ND2-Vn and either pcDNA3 or the NF-GFP-ND2-TM-6-8 construct. Data were normalized to average Venus fluorescence intensity determined for GluN1-positive HEK293 cells transfected with GluN1ΔCTD-Vc alone . The observed fluorescence intensity was significantly different between GluN1ΔCTD-Vc+ND2-Vn+pcDNA3 (3.05±0.31, n=29) and both GluN1ΔCTD-Vc+ND2-Vn+NF-GFP-ND2-TM-6-8 (1.62±0.16, n=19), and GluN1ΔCTD-Vc only (1.00±0.17, n=17). Statistically significant differences between populations are indicated by the symbols ‘***’ and ‘****’ (P<0.001 and P<0.0001, respectively), and were evaluated by Welch’s one-way ANOVA test. Results are presented as mean±s.e.m. (b) Immunocytochemically stained primary hippocampal neurons transfected with GFP-ND2-TM-6-8. Anti GFP, GluN1 and MAP2 antibodies were used for visualization. Scale bar, 10 μm. (c) Representative traces displaying NMDAR currents recorded in primary hippocampal neurons transfected with GFP+GFP-ND2-TM-6-8, GFP+GFP-ND2-TM-6-7 or GFP alone for 48 h. Src-activating peptide (EPQ(pY)EEIPIA) was included in the patch-recording electrode pipette. GFP was added in each case to monitor the successfully transfected neurons due to the relatively poorly fluorescing GFP-ND2 fusion proteins. GFP-ND2-TM-6-7 is known not to interact with GluN1, so was chosen as a control to monitor non-specific effects. (d) Grouped data from c. ND2-TM-6-8+GFP (n=14), ND2-TM-6-7+GFP (n=9) and GFP alone (n=6). Statistically significant differences between populations at 15 min are indicated by the symbol ‘*’ (P<0.05), and were evaluated by Kruskal–Wallis non-parametric analysis of variance with Dunn’s multiple post hoc comparison tests. ND2-TM-6-8+GFP (n=9), ND2-TM-6-7+GFP (n=6) and GFP alone (n=4) at 15th minute. n=# of cells. Results are presented as mean±s.e.m. See also Supplementary Fig. 13.