Figure 2
Schizophrenia and DD/ID-associated GRIN2A mutations demonstrate both gain- and loss-of-function effects. (A) Whole-cell recording protocol using Syncropatch to record NMDAR currents from HEK cells transiently transfected with the construct shown in Fig. 1B. Cells were puffed with different concentrations of glutamate stacked in between different volumes of reference solution (light gray triangles, inset on the right panel). Each puff results in a ~ 250 ms transient of glutamate exposure to the receptors. After each glutamate application half of the volume of the well was replaced with fresh reference solution (“Reference exchange”, gray bars) to minimize desensitization due to residual glutamate in the well. Glu, glutamate; ref, reference solution. (B) Averaged current traces of wild-type and selected mutant NMDARs evoked by ~ 250 ms transients of glutamate exposure of increasing concentration, in the presence of 30 μM glycine. The traces were colored according to the different concentrations of glutamate. (C) Averaged current density in response to increasing concentrations of glutamate in the constant presence of 30 μM glycine, normalized to maximal response, for wild-type and selected mutant NMDARs. The lines indicate a nonlinear regression three-parameter fit to each dataset. (D) Peak current density in response to 100 µM glutamate, normalized to wild type’s response, for each mutant NMDAR. (E) Glutamate EC50 normalized to wild type EC50, for each mutant NMDAR. In (C–E) data are displayed as mean ± SEM, n = 10-77; see Table 1 for number of cells recorded per variant; statistical significance was assessed using Brown-Forsythe and Welch ANOVA with Dunnett’s T3 multiple comparisons test. *: p < 0.05, ***: p < 0.001, ****: p < 0.0001.
