Structural features in the glycine-binding sites of the GluN1 and GluN3A subunits regulate the surface delivery of NMDA receptors

Kristyna Skrenkova,Martina Kaniakova,Stepan Kortus,Martin Horak,Katarina Hemelikova,Barbora Krausova,Marharyta Kolcheva

doi:10.1038/s41598-019-48845-3

Abstract

N-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that play an essential role in mediating excitatory neurotransmission in the mammalian central nervous system (CNS). Functional NMDARs are tetramers composed of GluN1, GluN2A-D, and/or GluN3A-B subunits, giving rise to a wide variety of NMDAR subtypes with unique functional properties. Here, we examined the surface delivery and functional properties of NMDARs containing mutations in the glycine-binding sites in GluN1 and GluN3A subunits expressed in mammalian cell lines and primary rat hippocampal neurons. We found that the structural features of the glycine-binding sites in both GluN1 and GluN3A subunits are correlated with receptor forward trafficking to the cell surface. In addition, we found that a potentially clinically relevant mutation in the glycine-binding site of the human GluN3A subunit significantly reduces surface delivery of NMDARs. Taken together, these findings provide novel insight into how NMDARs are regulated by their glycine-binding sites and may provide important information regarding the role of NMDARs in both physiological and pathophysiological processes in the mammalian CNS.

Highlights

N-methyl-D-aspartate receptors (NMDARs) are a subclass of glutamate receptors that play an essential role in synapse development, excitatory neurotransmission as well as synaptic plasticity in the mammalian central nervous system (CNS)[1,2]
We found that GluN1-4a/GluN3A-ΔCTD receptors are delivered to the surface at wild-type levels; in contrast, GluN1-4a/GluN3A-ΔATD + ΔS1 receptors failed to traffic to the cell surface (Fig. 1b,c)
We examined receptors containing GluN3A subunits that lack either the amino-terminal domain (ATD) (GluN3A-ΔATD) or the S1 segment (GluN3A-ΔS1; Fig. 1a) ‒ and found that GluN1-4a/GluN3A-ΔATD receptors are expressed at the cell surface, whereas GluN1-4a/GluN3A-ΔS1 receptors failed to traffic to the cell surface (Fig. 1b,c; see the Discussion)

Summary

Introduction

N-methyl-D-aspartate receptors (NMDARs) are a subclass of glutamate receptors that play an essential role in synapse development, excitatory neurotransmission as well as synaptic plasticity in the mammalian central nervous system (CNS)[1,2]. Whether structural changes in the glycine-binding sites in the GluN1 and/or GluN3A subunits regulate the surface delivery of functional GluN3A-containing NMDARs is currently unknown. We used a combination of microscopy, quantitative assays and electrophysiology in mammalian cell lines and primary rat hippocampal neurones in order to investigate whether disrupting the structure of the glycine-binding sites in the LBDs of GluN1 and/or GluN3A subunits affects the surface delivery of GluN3A-containing NMDARs. We found that mutant GluN1 and GluN3A subunits have reduced glycine sensitivity, and this reduction is correlated with reduced forward trafficking of NMDARs to the cell surface. We found that mutant GluN1 and GluN3A subunits have reduced glycine sensitivity, and this reduction is correlated with reduced forward trafficking of NMDARs to the cell surface These results were supported by additional experiments using the human GluN3A subunit. Our results provide novel insight into the mechanisms that regulate the number, type, and function of NMDARs at the neuronal surface

Methods

Results

Conclusion