Figure 6

A714L mutation in GluN1 prevents glycine primed NMDAR internalization. A, Left, Traces show responses to the test applications recorded from cells expressing GluN1/GluN2B (upper) or GluN1.A714L/GluN2B (lower). Times indicates time after glycine treatment (10 mM, 5 min); t = 0 is the first response after glycine treatment. Right, Normalized NMDA currents (I/I0) vs time of the experiment from cells expressing GluN1/GluN2B (n = 4 cells) or GluN1.A714L/GluN2B (n = 3 cells) receptors. Glycine (10 mM, 5 min) was applied during the period indicated by the bar above the graph. B, NMDAR internalization measured by cell ELISA assay using HEK293 cells expressing GluN1.A714L/GluN2B receptors (n = 6). * indicates p < 0.05 compared to ECS control. C, Cells expressing BBS-GluN1.A714L/GluN2B showed no internalization as measured by CypHer5E fluorescence in cells. Scale bar = 10 μm D, Top, Representative Western blot of co-immunoprecipitation of NMDARs with adaptin β2 from cell lysate treated with glycine (10 mM) plus APV (100 μM) or APV (100 μM) alone. Bottom, Quantification is summarized in the histogram showing mean GluN1-adaptin β2 association from HEK293 cells expressing GluN1/GluN2B and GluN1.A714L/GluN2B receptors after glycine treatment (n = 10). Data are presented as mean percent (± s.e.m.) of APV treated group. Data were normalized to amount of adaptin β2 immunoprecipitated. ** indicates p < 0.01 compared to control.