N-methyl-D-aspartate receptors (NMDARs) play an essential role in excitatory neurotransmission in the mammalian brain, and their physiological importance is underscored by the large number of pathogenic mutations that have been identified in the receptor's GluN subunits and associated with a wide range of diseases and disorders. Here, we characterized the functional and pharmacological effects of the pathogenic N650K variant in the GluN1 subunit, which is associated with developmental delay and seizures. Our microscopy experiments showed that when expressed in HEK293 cells (from ATCC®), the GluN1-N650K subunit increases the surface expression of both GluN1/GluN2A and GluN1/GluN2B receptors, but not GluN1/GluN3A receptors, consistent with increased surface expression of the GluN1-N650K subunit expressed in hippocampal neurons (from embryonic day 18 of Wistar rats of both sexes). Using electrophysiology, we found that the GluN1-N650K variant increases the potency of GluN1/GluN2A receptors to both glutamate and glycine but decreases the receptor's conductance and open probability. In addition, the GluN1-N650K subunit does not form functional GluN1/GluN2B receptors but does form fully functional GluN1/GluN3A receptors. Moreover, in the presence of extracellular Mg2+, GluN1-N650K/GluN2A receptors have a similar and increased response to ketamine and memantine, respectively, while the effect of both drugs had markedly slower onset and offset compared to wild-type GluN1/GluN2A receptors. Finally, we found that expressing the GluN1-N650K subunit in hippocampal neurons reduces excitotoxicity, and memantine shows promising neuroprotective effects in neurons expressing either wild-type GluN1 or the GluN1-N650K subunit. This study provides the functional and pharmacological characterization of NMDARs containing the GluN1-N650K variant.

Department of Molecular Biology and Biochemistry Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Flemingovo namesti 542 2 P O Box 16000 Prague 6 Czech Republic

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

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; Laboratory of Cellular Neurophysiology Institute of Physiology of the Czech Academy of Sciences Videnska 1083 14220 Prague 4 Czech Republic

References provided by Crossref.org

Distinct Regulation of Early Trafficking of the NMDA Receptors by the Ligand-Binding Domains of the GluN1 and GluN2A Subunits

Analysis of Surface Expression of NMDAR Subunits in Primary Hippocampal Neurons

Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology

Subunit-Dependent Surface Mobility and Localization of NMDA Receptors in Hippocampal Neurons Measured Using Nanobody Probes