Skrenkova Kristyna, Lee Sanghyeon, Lichnerova Katarina, Kaniakova Martina, Hansikova Hana, Zapotocky Martin, Suh Young Ho, Horak Martin
Department of Cellular Neurophysiology, Institute of Physiology of the Czech Academy of Sciences, Prague, Czechia.
Department of Neurochemistry, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia.
Front Mol Neurosci. 2018 Jun 4;11:188. doi: 10.3389/fnmol.2018.00188. eCollection 2018.
-methyl-D-aspartate receptors (NMDARs) play critical roles in both excitatory neurotransmission and synaptic plasticity. NMDARs containing the nonconventional GluN3A subunit have different functional properties compared to receptors comprised of GluN1/GluN2 subunits. Previous studies showed that GluN1/GluN2 receptors are regulated by -glycosylation; however, limited information is available regarding the role of -glycosylation in GluN3A-containing NMDARs. Using a combination of microscopy, biochemistry, and electrophysiology in mammalian cell lines and rat hippocampal neurons, we found that two asparagine residues (N203 and N368) in the GluN1 subunit and three asparagine residues (N145, N264 and N275) in the GluN3A subunit are required for surface delivery of GluN3A-containing NMDARs. Furthermore, deglycosylation and lectin-based analysis revealed that GluN3A subunits contain extensively modified -glycan structures, including hybrid/complex forms of -glycans. We also found (either using a panel of inhibitors or by studying human fibroblasts derived from patients with a congenital disorder of glycosylation) that -glycan remodeling is not required for the surface delivery of GluN3A-containing NMDARs. Finally, we found that the surface mobility of GluN3A-containing NMDARs in hippocampal neurons is increased following incubation with 1-deoxymannojirimycin (DMM, an inhibitor of the formation of the hybrid/complex forms of -glycans) and decreased in the presence of specific lectins. These findings provide new insight regarding the mechanisms by which neurons can regulate NMDAR trafficking and function.
N-甲基-D-天冬氨酸受体（NMDARs）在兴奋性神经传递和突触可塑性中均发挥关键作用。与由GluN1/GluN2亚基组成的受体相比，含有非常规GluN3A亚基的NMDARs具有不同的功能特性。先前的研究表明，GluN1/GluN2受体受N-糖基化调节；然而，关于N-糖基化在含GluN3A的NMDARs中的作用，可用信息有限。通过在哺乳动物细胞系和大鼠海马神经元中结合使用显微镜、生物化学和电生理学方法，我们发现GluN1亚基中的两个天冬酰胺残基（N203和N368）以及GluN3A亚基中的三个天冬酰胺残基（N145、N264和N275）是含GluN3A的NMDARs向细胞表面转运所必需的。此外，去糖基化和基于凝集素的分析表明，GluN3A亚基含有广泛修饰的N-聚糖结构，包括杂合/复杂形式的N-聚糖。我们还发现（无论是使用一组抑制剂还是通过研究来自先天性糖基化障碍患者的人成纤维细胞），含GluN3A的NMDARs向细胞表面转运不需要N-聚糖重塑。最后，我们发现用1-脱氧甘露基野尻霉素（DMM，一种N-聚糖杂合/复杂形式形成的抑制剂）孵育后，海马神经元中含GluN3A的NMDARs的表面流动性增加，而在存在特定凝集素的情况下则降低。这些发现为神经元调节NMDAR转运和功能的机制提供了新的见解。
