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.
Highlights
(NR2A-D) encoded by different but closely related genes
Our data showed that NR subunits ( NR1–1a and NR2A and NR2B) formed dimers in living cells before they formed functional NMDA receptors
Evidence from the blue native PAGE system and sucrose-gradient centrifugation analysis suggests that the NR1 subunits expressed alone in heterogeneous cells can self-associate to form homodimers, whereas the NR2 subunits may not form homo-oligomers similar to the NR1–1a subunits [26], the current model of NMDA receptor arrangement is a dimer of dimers based on the tandem and truncated experiment [28]
Summary
(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. 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 [13,14,15]. 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). Biochemical studies indicate that the NR1–1a subunit expressed alone can form homodimers, and the first 380 amino acids in the N terminus are important for the association of NR1–1a with NR2A [26]. FRET Analysis of NMDA Receptor Assembly ings indicate that dimerization may be a general principle guiding the assembly of NMDA receptors, as well as AMPA receptors (29 –31), Kϩ channels [39], and cyclic nucleotidegated channels [40]
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