Figure 4.

GluN2A^N615K reduces Mg²⁺ blockade and current density in cultured neurons

A–C, representative whole‐cell, voltage clamp recordings made from DIV 9 primary mouse cortical neurons transiently transfected with an inert control: β globin (A), GluN2A^WT (B) or GluN2A^N615K (C). Traces show the response to saturating NMDA (150 μm) and inhibition by Mg²⁺ (1 mm), before and after a 1 min application of ifenprodil (3 μm) (a selective GluN2B negative allosteric modulator). D, summary data showing percentage inhibition by ifenprodil. A one‐way ANOVA showed a significant effect of transfected subunit (F _2,42 = 46, P = 2.8 × 10^–11), with post hoc Bonferroni corrected t tests indicating that neurons transfected with GluN2A^WT showed lower ifenprodil sensitivity than control transfection cells (WT: 32 ± 3; n = 15; globin 75 ± 2; n = 15; t _21.9 = 10.7, P = 1.0 × 10^–9), as did neurons transfected with GluN2A^N615K (43 ± 4; n = 15; t _20.0 = 7.2, P = 1.9 × 10^–6) and with no difference between GluN2A and GluN2A^N615K (t _27.3 = 1.9, P = 0.19). These results confirm that the GluN2A^N615K subunits have been successfully trafficked to the surface in neurons. E, summary data showing Mg²⁺ blockade in the presence and absence of ifenprodil. Squares show blockade without ifenprodil; triangles show blockade with ifenprodil. Solid shapes represent means; hollow shapes are individual cells. A two‐way ANOVA showed a significant effect of transfected subunit (F _2,42 = 111, P = 2 × 10^–16) and the presence of ifenprodil (F _1,42 = 31, P = 1.9 × 10^–6), with a significant interaction (F _2,42 = 90, P = 6.7 × 10^–16). Post hoc Bonferroni corrected t tests showed lower Mg²⁺ blockade in the absence of ifenprodil in neurons transfected with GluN2A^N615K compared to GluN2A^WT (WT: 93 ± 1%; n = 15; N615K 52 ± 5%; n = 15; t _16.7 = 8.2, P = 9.7 × 10^–7), although there was no difference between GluN2A^WT and control (globin: 97 ± 1%; n = 15; t _23.3 = 2.2, P = 0.13). In the presence of ifenprodil, the reduction in Mg²⁺ blockade associated with GluN2A^N615K vs. wild‐type was even more marked (WT: 95 ± 1%; n = 15; N615K 31 ± 5%; n = 15; t _15.4 = 12.1, P = 8.4 × 10^–9). F, summary data showing current density in the presence and absence of ifenprodil. As with Mg²⁺ blockade, we found that current density was reduced in neurons transfected with GluN2A^N615K, and this effect was more pronounced in the presence of ifenprodil. A two‐way ANOVA showed a significant effect of transfected subunit (F _2,42 = 9.4, P = 0.0004) and the presence of ifenprodil (F _1,42 = 178, P = 2 × 10^–16), with a significant interaction (F _2,42 = 9.2, P = 0.0005). Post hoc Bonferroni corrected t tests showed lower current density in the absence of ifenprodil in neurons transfected with GluN2A^N615K compared to GluN2A^WT (WT: 58 ± 5 pA pF^–1; n = 15; N615K 40 ± 4 pA pF^–1; n = 15; t _27.3 = 2.7, P = 0.037), although there was no difference between GluN2A^WT and control (globin: 45 ± 4 pA pF^–1; n = 15; t _27.4 = 1.9, P = 0.22). In the presence of ifenprodil, the reduction in current density associated with GluN2A^N615K vs. wild‐type was even more marked (WT: 38 ± 4 pA pF^–1; n = 15; N615K 23 ± 3 pA pF^–1; n = 15) (t _27.8 = 3.3, P = 0.009).