Figure 5

Mutant GluN1.RAAL/GluN2B receptors did not show glycine priming. A, Left, Traces show individual responses to the test applications recorded from cells expressing GluN1/GluN2B (upper) or GluN1.RRAL/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 from cells expressing GluN1/GluN2B (n = 4 cells) or GluN1.RRAL/GluN2B (n = 9 cells). Glycine (10 mM, 5 min) was applied during the period indicated by the bar above the graph. B, NMDAR internalization as quantified by cell ELISA assay using HEK293 cells expressing GluN1/GluN2B or GluN1.RRAL/GluN2B receptors. Cultures (n = 6) were pre-treated with ECS or ECS containing glycine (10mM) plus APV (100 μM) followed by ECS or with NMDA (50 μM) plus glycine (100 μM). * indicates p < 0.05 compared with ECS control. C, Cells expressing BBS-GluN1.RRAL/GluN2B showed minimal CypHer5E fluorescence after treatment of NMDA (50 μM) plus glycine (100 μM) with 10 mM glycine plus 100 μM APV pre-treatment. 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 was summarized in the histogram showing mean GluN1-adaptin β2 association from HEK293 cells expressing GluN1/GluN2B and GluN1.RRAL/GluN2B receptors after glycine treatment (n = 10). Data are presented as mean percent (± s.e.m.) of APV treated group. Data were normalized to the amount of adaptin β2 immunoprecipitated. ** indicates p < 0.01 compared to control.