N-methyl-D-aspartate receptors (NMDARs) are a family of ionotropic glutamate receptors that mediate excitatory neurotransmission in the CNS. Native NMDARs are assembled as heterotetramers composed of two obligatory GluN1 and two GluN2A-D/GluN3A-B subunits at various combinations. The opening of the ion channel is a central feature of NMDAR function and is controlled by binding of two co-agonists (glutamate/glycine) to the agonist binding domains (ABD). In our previous work, using molecular dynamics simulations, we proposed functionally important residues in this process of channel opening, Glu522 and Arg695 in GluN1, and Asp786 in GluN2B surrounded by two conserved glycines (termed us as GDG motif). Here, we aimed to assess this in silico data to confirm specific role of GDG during the process of channel opening. Therefore, we prepared the constructs where identified residues were mutated in order to eliminate or impair presumed intramolecular interactions. Using electrophysiology techniques we have revealed that NMDARs with mutated GluN2A/B subunits have slower time-course of MK-801 block compared to WTs, with the average Tauw 2990ms and 897ms respectively. Mutated GluN2A/B subunits also exhibited lower affinity to agonists and affected the receptor inhibition of ABD modulator TCN-201 with average inhibition 35% compared to 78% in WT. Additionally, immunocytochemical staining of the NMDAR surface-expression in the HEK293 cells showed that presence of altered GDG motif significantly decreased the surface expression of NMDARs. These results showed the role of GDG motif in NMDAR localization and function, confirming the intrasubunit interactions in the open state of the NMDAR.