The Role of Disulfide Bonds in the GluN1 Subunit in the Early Trafficking and Functional Properties of GluN1/GluN2 and GluN1/GluN3 NMDA Receptors

J Neurosci. 2026 Apr 1;46(13):e0409252026. doi: 10.1523/JNEUROSCI.0409-25.2026.

Abstract

N-Methyl-d-aspartate receptors (NMDARs) are ionotropic glutamate receptors essential for excitatory neurotransmission. Previous studies proposed the existence of four disulfide bonds in the GluN1 subunit; however, their role in NMDAR trafficking remains unclear. Our study first confirmed the existence of four disulfide bonds in the GluN1 subunit using biochemistry in human embryonic kidney 293T (HEK293T) cells. Disrupting the individual disulfide bonds by serine replacements produced the following surface expression trend for GluN1/GluN2A, GluN1/GluN2B, and GluN1/GluN3A receptors: wild-type (WT) > GluN1-C744S-C798S > GluN1-C79S-C308S > GluN1-C420S-C454S > GluN1-C436S-C455S subunits. Electrophysiology revealed altered functional properties of NMDARs with disrupted disulfide bonds, specifically an increased probability of opening (Po) at the GluN1-C744S-C798S/GluN2 receptors. Synchronized release from the endoplasmic reticulum confirmed that disruption of disulfide bonds impaired early trafficking of NMDARs in HEK293T cells and primary hippocampal neurons prepared from Wistar rats of both sexes (Embryonic Day 18). The pathogenic GluN1-C744Y variant, associated with neurodevelopmental disorder and seizures, caused reduced surface expression and increased Po at GluN1/GluN2 receptors, consistent with findings for the GluN1-C744S-C798S subunit. The FDA-approved memantine inhibited GluN1-C744Y/GluN2 receptors more potently and with distinct kinetics compared with WT GluN1/GluN2 receptors. We also observed enhanced NMDA-induced excitotoxicity in hippocampal neurons expressing the GluN1-C744Y subunit, which memantine reduced more effectively compared with the WT GluN1 subunit. Lastly, we demonstrated that the presence of the hGluN1-1a-C744Y subunit counteracted the effect of the hGluN3A subunit on decreasing dendritic spine maturation, consistent with the reduced surface delivery of the NMDARs carrying this variant.

Keywords: Golgi apparatus; endoplasmic reticulum; excitotoxicity; glutamate receptor; ion channel; pathogenic variant.

MeSH terms

  • Animals
  • Cells, Cultured
  • Disulfides* / metabolism
  • Female
  • HEK293 Cells
  • Hippocampus / cytology
  • Hippocampus / metabolism
  • Humans
  • Male
  • Nerve Tissue Proteins* / genetics
  • Nerve Tissue Proteins* / metabolism
  • Neurons / drug effects
  • Neurons / metabolism
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Rats
  • Receptors, N-Methyl-D-Aspartate* / chemistry
  • Receptors, N-Methyl-D-Aspartate* / genetics
  • Receptors, N-Methyl-D-Aspartate* / metabolism

Substances

  • Receptors, N-Methyl-D-Aspartate
  • Disulfides
  • GRIN1 protein, human
  • Nerve Tissue Proteins