Mutations in the glycine-binding site in the GluN3A subunit regulate the surface expression and glycine affinity of GluN1/GluN3A receptors. (a) Schematic representation of the glycine-binding site in the GluN3A subunit (PDB code: 2RC7; in orange), with a glycine molecule shown; for comparison, the binding site in the GluN1 subunit is shown in blue. (b) Representative images of COS-7 cells transfected with the indicated wild-type or mutant GFP-GluN3A subunit together with GluN1-4a subunit and labelled 24 h after transfection. (c) Summary of the relative surface expression of the indicated GluN subunits measured using fluorescence microscopy (n ≥ 20 cells per group); *p < 0.05 vs. GluN1-4a/GluN3A (ANOVA). (d) Heterologous HEK293 cells co-expressing the indicated GluN1-4a and GFP-GluN3A (GluN3A) subunits were labeled with primary anti-GFP and secondary antibodies in non-permeabilizing and permeabilizing conditions. The bar graphs show quantification of relative surface expression of the indicated GluN subunit combinations obtained using quantitative colorimetric assay (n = 6); *p < 0.05 relative to GluN1-4a/GluN3A (ANOVA). (e,h) Representative whole-cell patch-clamp recordings from HEK293 cells transfected with the indicated wild-type or mutant GluN1-4a/GluN3A receptors. Currents were elicited by applying the indicated concentrations of glycine. (f) Summary of the τw of desensitisation for the indicated GluN1-4a/GluN3A (n ≥ 5 cells per group). (g) Peak concentration-response curves for the indicated wild-type and mutant GluN1-4a/GluN3A receptors. Each data point represents the mean relative current from ≥5 cells. The EC50 values and Hill coefficients are listed in Table 1. (i) The pharmacological analysis with CGP-78608 at the GluN1/GluN3A receptors. Representative whole-cell patch-clamp recordings from HEK293 cells transfected with the indicated wild-type or mutant GluN1-4a/GluN3A receptors at a membrane potential of −60 mV. Currents were elicited by applying 1,000 µM glycine; 0.5 µM CGP78608 was applied as indicated. (j) Summary of current densities (pA/pF) obtained from the HEK293 cells expressing the indicated GluN1-4a/GluN3A receptors (n ≥ 6 cells per group).