N-Glycosylation Regulates the Trafficking and Surface Mobility of GluN3A-Containing NMDA Receptors.

Kristyna Skrenkova,Sanghyeon Lee,Martin Horak,Young Ho Suh,Hana Hansikova,Martin Zapotocky,Katarina Lichnerova,Martina Kaniakova

doi:10.3389/fnmol.2018.00188

Abstract

N-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 N-glycosylation; however, limited information is available regarding the role of N-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 N-glycan structures, including hybrid/complex forms of N-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 N-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 N-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.

Highlights

N-methyl-D-aspartate receptors (NMDARs), a subclass of glutamate receptors, are essential for excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS; Traynelis et al, 2010; Horak et al, 2014)
We first examined whether specific N-glycosylation site(s) in GluN1 and/or GluN3A are required for the trafficking of GluN3A-containing NMDARs to the cell surface
Our electrophysiological measurements in transfected human embryonic kidney 293 (HEK293) cells showed that co-expressing the GluN1–4a splice variant with GluN3A produces larger glycineinduced currents compared to co-expressing the GluN1–1a subunit; this finding is consistent with previous reports (Smothers and Woodward, 2009; Cummings and Popescu, 2016)

Summary

Introduction

N-methyl-D-aspartate receptors (NMDARs), a subclass of glutamate receptors, are essential for excitatory neurotransmission and synaptic plasticity in the mammalian central nervous system (CNS; Traynelis et al, 2010; Horak et al, 2014). Many studies have examined the molecular mechanisms that regulate the surface expression and surface mobility of NMDARs, these studies focused largely on NMDAR subtypes containing GluN1/GluN2 subunits (Traynelis et al, 2010; Sanz-Clemente et al, 2012). GluN1/GluN2 receptors have been found to be extensively N-glycosylated in both heterologous expression systems (Chazot et al, 1995; Everts et al, 1997) and native preparations (Huh and Wenthold, 1999; Kaniakova et al, 2016); we recently reported that two conventional N-glycosylation sites in the GluN1 subunit are required for the release of GluN1/GluN2 receptors from the endoplasmic reticulum (ER; Lichnerova et al, 2015); the role of N-glycosylation in the trafficking and/or surface mobility of NMDARs containing the nonconventional GluN3A subunit has not been examined

Methods

Results

Conclusion