AIMS:Investigate whether the endoplasmic reticulum (ER) quality control machinery verifies the structure of ligand-binding domains (LBDs) of NMDARs or controls the functionality of NMDARs.METHODS:HEK293 cells were used to express wild-type (WT) or mutation forms of GluN1/GluN2A and GluN1/GluN2B receptors. Surface expressions were observed using fluorescence confocal microscopy. Electrophysiological properties of NMDARs were assessed via the whole-cell patch-clamp technique.RESULTS:Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A and GluN1/GluN2B receptors strongly correlates with EC50 values for glycine and L-glutamate. Interestingly, co-expression of both GluN1 and GluN2A subunits with alanine substitutions led to an additive reduction in the surface number of GluN1/GluN2A receptors. Furthermore, the human versions of GluN1/GluN2A receptors containing pathogenic GluN1-S688Y, GluN1-S688P, GluN1-D732E, GluN2A-S511L, and GluN2A-T690M variants exhibited distinct surface expression compared to the corresponding alanine substitutions. Mutation cycles involving GluN1-S688, GluN1-D732, GluN2A-S511, and GluN2A-T690 residues revealed, in most cases, a weak correlation between surface expression of the mutant GluN1/GluN2A receptors and their EC50 values for glycine or L-glutamate.CONCLUSIONS:This study indicates that the ER quality control machinery perceives structural changes in the LBDs but not the functionality of GluN1/GluN2A and GluN1/GluN2B receptors.ACKNOWLEDGMENTS:This work was supported by the Czech Science Foundation (24-10026S), project registration number LX22NPO5107 (MEYS CR): Financed by EU – Next Generation EU and by project registration number CZ.02.01.01/00/22_008/0004562 (ExRegMed, MEYS CR).