AIMS: Investigate the impact of disrupting specific disulfide bonds in the GluN1 subunit on the early trafficking and functional properties of unconventional NMDARs.METHODS:HEK293 cells and rat hippocampal neurons were used to express wild-type (WT) or mutant forms of GluN1/GluN3A receptors. Surface expression and colocalization of NMDARs with the Golgi apparatus (GA) were observed using fluorescence confocal microscopy. Electrophysiological properties of NMDARs were assessed via patch-clamp technique.RESULTS:Disrupting disulfide bonds in the GluN1 subunit co-transfected with GluN3A subunit led to varying levels of surface expression in HEK293 cells, with a trend observed: WT > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S. Microscopic data using ARIAD technology, which allows controlled secretion from the endoplasmic reticulum, showed the following order of co-localization with GA: WT ARIAD-GluN1 > ARIAD-GluN1-C744S-C798S > ARIAD-GluN1-C79S-C308S > ARIAD-GluN1-C420S-C454S > ARIAD-GluN1-C436S-C455S. These experiments support the hypothesis that the downregulation of surface expression of NMDARs with disrupted disulfide bonds occurs at the level of early trafficking, likely during ER processing. Electrophysiological analysis revealed that disruption of disulfide bond GluN1-C744S-C798S increased desensitization and EC50 values for glycine. The potential pathogenic variant GluN1-C744Y co-transfected with GluN3A reduced the surface expression of NMDARs to ~50 % in hippocampal neurons.CONCLUSIONS: Disruption of disulfide bonds in the GluN1 subunit and the pathogenic variant GluN1-C744Y have notable effects on surface expression and electrophysiological properties in unconventional diheteromeric NMDARs.This work was supported by a project from the Grant Agency of Charles University (GAUK: 252314) and by project registration number CZ.02.01.01/00/22_008/0004562 (Exregmed, MEYS CR).