Although numerous pathogenic mutations have been identified in various subunits of N-methyl-D-aspartate receptors (NMDARs), ionotropic glutamate receptors that are central to glutamatergic neurotransmission, the functional effects of these mutations are often unknown. Here, we combined in silico modelling with microscopy, biochemistry, and electrophysiology in cultured HEK293 cells and hippocampal neurons to examine how the pathogenic missense mutation S688Y in the GluN1 NMDAR subunit affects receptor function and trafficking. We found that the S688Y mutation significantly increases the EC50 of both glycine and D-serine in GluN1/GluN2A and GluN1/GluN2B receptors, and significantly slows desensitisation of GluN1/GluN3A receptors. Moreover, the S688Y mutation reduces the surface expression of GluN3A-containing NMDARs in cultured hippocampal neurons, but does not affect the trafficking of GluN2-containing receptors. Finally, we found that the S688Y mutation reduces Ca2+ influx through NMDARs and reduces NMDA-induced excitotoxicity in cultured hippocampal neurons. These findings provide key insights into the molecular mechanisms that underlie the regulation of NMDAR subtypes containing pathogenic mutations.

Biomedical Research Centre University Hospital Hradec Kralove Sokolska 581 500 05 Hradec Kralove Czech Republic

Department of Biomedical Sciences Neuroscience Research Institute Seoul National University College of Medicine 103 Daehak ro Jongno gu Seoul 03080 South Korea

Department of Military Medical Service Organisation and Management and Department of Toxicology and Military Pharmacy; Faculty of Military Health Sciences University of Defence Trebesska 1575 500 01 Hradec Kralove Czech Republic

Department of Neurochemistry Institute of Experimental Medicine of the Czech Academy of Sciences Videnska 1083 14220 Prague 4 Czech Republic

Department of Physiology Faculty of Science Charles University Prague Albertov 6 12843 Prague 2 Czech Republic

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