Subunit Assembly of N-Methyl-d-aspartate Receptors Analyzed by Fluorescence Resonance Energy Transfer

Abstract

N-methyl-D-aspartate (NMDA) receptors play major roles in synaptic transmission and plasticity, as well as excitotoxicity. NMDA receptors are thought to be tetrameric complexes mainly composed of NMDA receptor (NR)1 and NR2 subunits. The NR1 subunits are required for the formation of functional NMDA receptor channels, whereas the NR2 subunits modify channel properties. Biochemical and functional studies indicate that subunits making up NMDA receptors are organized into a dimer of dimers, and the N termini of the subunits are major determinants for receptor assembling. Here we used a biophysical approach, fluorescence resonance energy transfer, to analyze the assembly of intact, functional NMDA receptors in living cells. The results showed that NR1, NR2A, and NR2B subunits could form homodimers when they were expressed alone in HEK293 cells. Subunit homodimers were also found existing in heteromeric NMDA receptors formed between NR1 and NR2 subunits. These findings are consistent with functional NMDA receptors being arranged as a dimer of dimers. In addition, our data indicated that the conformation of NR1 subunit homodimers was affected by the partner NR2 subunits during the formation of heteromeric receptor complexes, which might underlie the mechanism by which NR2 subunits modify NMDA receptor function. N-Methyl-D-aspartate (NMDA)1 receptors are a subtype of the ionotropic glutamate receptors, which also include ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors and kainate receptors. NMDA receptors play critical roles in synaptic plasticity, memory formation, and several types of neurological disorders (1-3). Three gene families, named NR1, NR2, and NR3, encode subunits composing NMDA receptors. The NR1 subunit is encoded by a single gene, which undergoes extensive splicing to yield eight splice variants, NR1-1a and -1b to NR1-4a and -4b. The NR2 subunit exists in four isoforms (NR2A-D) encoded by different but closely related genes (reviewed in Ref.2). All of the subunits have a similar membrane topological structure, with three transmembrane domains plus a loop region, an extracellular N terminus, and an intracellular C terminus. Recently, NMDA receptors were thought to be tetrameric channels, for which the relevant evidence comes from the electrophysiological studies of NMDA and AMPA receptors (4 -6) and the evolutionary link between glutamate receptors and tetrameric K ϩ channels (7, 8). The NR1 subunit contains a glycine-binding site (9, 10) and is essential for the formation of functional NMDA receptor channels, whereas the NR2 subunit provides a glutamate-binding site (11, 12) and modifies channel properties, such as current kinetics and channel conductance (13-15). The NR3 subunit does not form a functional NMDA receptor alone, but it can co-assemble with NR1/NR2 complexes to modulate the activity of the NMDA receptor (16 -18).Previous studies have shown that NMDA receptor complexes may contain different NR1 splice variants (19,20) or different NR2 subunits (21-24). Several distinct NMD...

Excitotoxicity and focal cerebral ischemia induce truncation of the NR2A and NR2B subunits of the NMDA receptor and cleavage of the scaffolding protein PSD-95

“…24 Plasmids pME18S-HA-NR2A and pME18S-HA-NR2B were generous gifts from Dr T Yamamoto (University of Tokyo, Tokyo, Japan) and contain, respectively, the rat NR2A and the mouse NR2B cDNAs with hemagglutinin epitopes in the N terminus between amino-acid residues 51 and 52 for NR2A 25 and between residues 66 and 67 for NR2B. 26 The Syn-GFP lentivirus plasmid was used as a vector for NR2A and NR2B sub-cloning and to produce virus.…”

Clustering of surface NMDA receptors is mainly mediated by the C-terminus of GluN2A in cultured rat hippocampal neurons

“…C o n s t r u c t i o n o f E G F P -G l u N 2 B , E G F P -G l u N 2 A , ECFP-GluN2A, and ECFP-GluN2B was as described previously [5,15] . GFP or CFP was tagged to GluN2B or GluN2A at the extracellular N-terminus.…”

“…Functional NMDARs are believed to be tetrameric complexes assembled from two GluN1 and two GluN2 (GluN2A-2D) subunits [4,5] . Different NMDAR subtypes have distinct channel properties, such as open probability and time-course of currents [6] .…”

A Three Amino Acid Tail Following the TM4 Region of the N-Methyl-D-aspartate Receptor (NR) 2 Subunits Is Sufficient to Overcome Endoplasmic Reticulum Retention of NR1-1a Subunit

“…Average background signal was determined as the mean fluorescence intensity from an area not expressing the constructs and was subtracted from the raw images before carrying out FRET calculations. The effective FRET efficiency was calculated using the FRET ratio (FR) measurement (22)(23)(24) in Equation 1,…”