AIMS: Investigate the impact of disrupting specific disulfide bonds in the GluN1 subunit on the early trafficking and functional properties of conventional NMDARs.METHODS:HEK293 cells were utilized to express wild-type (WT) or mutant forms of GluN1/GluN2A and GluN1/GluN2B receptors. Surface expression and colocalization of NMDAR with the Golgi apparatus were observed using fluorescence confocal microscopy. Electrophysiological properties of NMDARs were assessed via the whole-cell patch-clamp technique. In addition, NMDA-induced excitotoxicity in rat hippocampal neurons was examined specifically for the GluN1-C744Y mutation.RESULTS:Disrupting disulfide bonds in the GluN1 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. Electrophysiological analysis revealed that disrupted disulfide bonds correlated with changes in EC50 values for L-glutamate and glycine but not with open probability (Po). The potential pathogenic variant GluN1-C744Y in combination with GluN2A or GluN2B increased Po ~2-3-fold and reduced to ~50% the surface expression of NMDAR. Neurons infected with the GluN1-C744Y mutation exhibited increased susceptibility to cell death at NMDA concentrations of 10 and 30 μM, consistent with the observed increase in Po for GluN1-C744Y/GluN2 combinations.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 conventional diheteromeric NMDAR.Suported by: Grant Agency of Charles University (GAUK: 252314), project registration number CZ.02.01.01/00/22_008/0004562 (Exregmed, MEYS CR). We acknowledge the Microscopy Service Centre (Institute of Experimental Medicine CAS) supported by the MEYS CR (LM2023050; Czech-Bioimaging).