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

Abstract

N-methyl-D-aspartate receptors (NMDARs) play a crucial role in excitatory neurotransmission, with numerous pathogenic variants identified in the GluN subunits, including their ligand-binding domains (LBDs). The prevailing hypothesis postulates that the endoplasmic reticulum (ER) quality control machinery verifies the agonist occupancy of NMDARs, but this was tested in a limited number of studies. Using microscopy and electrophysiology in the HEK293 cells, we found that surface expression of GluN1/GluN2A receptors containing a set of alanine substitutions within the LBDs correlated with the measured EC₅₀ values for glycine (GluN1 subunit mutations), while did not correlate with the measured EC₅₀ values for L-glutamate (GluN2A subunit mutations). The mutant cycle of GluN1-S688 residue, including the pathogenic GluN1-S688Y and GluN1-S688P variants, showed a correlation between relative surface expression of the GluN1/GluN2A receptors and the measured EC₅₀ values for glycine, as well as with the calculated ΔG_binding values for glycine obtained from molecular dynamics (MD) simulations. In contrast, the mutant cycle of GluN2A-S511 residue did not show any correlation between the relative surface expression of the GluN1/GluN2A receptors and the measured EC₅₀ values for L-glutamate or calculated ΔG_binding values for L-glutamate. Co-expression of both mutated GluN1 and GluN2A subunits led to additive or synergistic alterations in the surface number of GluN1/GluN2A receptors. The synchronized ER release by ARIAD technology confirmed the altered early trafficking of GluN1/GluN2A receptors containing the mutated LBDs. The microscopical analysis from embryonal rat hippocampal neurons (both sexes) corroborated our conclusions from the HEK293 cells.Significant statement We examined >80 mutant GluN1/GluN2 receptor combinations, including pathogenic and potentially pathogenic variants in the LBDs of GluN1 and GluN2A subunits. The combination of the experimentally measured (relative surface expression, EC₅₀ values) and calculated (ΔG_binding values, RMSD, and SASA) parameters revealed that the LBDs of the GluN1 and GluN2A subunits distinctly regulate the early trafficking of GluN1/GluN2A receptors. In addition, we validated a novel system of synchronized release of GluN1/GluN2A receptors from the ER. Our findings support the urgency of further detailed research on the regulation of early trafficking of NMDARs, as it may open the avenue to targeted intervention of central nervous system disorders associated with pathogenic variants in GluN subunits.