Independent regulation of early trafficking of NMDA receptors by ligand-binding domains of the GluN1 and GluN2A subunits

The essential role of N-methyl-D-aspartate receptors (NMDARs) in excitatory neurotransmission is underscored by numerous pathogenic variants in the GluN subunits, including those identified in their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs; however, whether it controls the structure of LBDs or the functionality of NMDARs is unknown. Using alanine substitutions combined with microscopy and electrophysiology, we found that surface expression of GluN1/GluN2A receptors, the primary NMDAR subtype in the adult forebrain, strongly correlates with EC ₅₀ 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, as did co-expression of both GluN1 and GluN2A subunits containing closed cleft conformation of LBDs. The synchronized ER release confirmed the altered regulation of early trafficking of GluN1/GluN2A receptors bearing alanine substitutions in the LBDs. 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. Mutant cycles of 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 EC ₅₀ values for glycine or L-glutamate. Consistent with our experimental data, molecular modeling and dynamics showed that the ER quality control machinery likely perceives structural changes of the LBDs but not the functionality of GluN1/GluN2A receptors.