Molecular Mechanisms Of Subunit Assembly, Trafficking And Surface Expression Of NMDA Receptors

N-methyl-D-aspartate (NMDA) receptors, which belong to the ionotropic glutamate receptors, play major roles in synaptic transmission and plasticity, as well as excitotoxicity. Different from non-NMDA receptors, NMDA receptors are thought to be hetero-tetrameric complex mainly composed of NR1 and NR2 subunits. When expressed alone in heterogenous cells, such as HEK293 cells, the NR subunit cannot egree the ER and express in the cell membrane. The NR1 subunits have eight splice variants and are required for the formation of functional NMDA receptor channels, while the NR2 subunits, containing four different genes NR2A-2D, mainly modify channel properties. The existence of many isoforms and subunits, and subtype-specific assembly create receptor functional heterogeneity, which in some degree determine the variable and complicated biological properties that NMDA receptors have in the brain. Despite the important findings obtained with the functional properties, the mechanism of NMDA receptor assembly remains more of a mystery. Especially as to the subunit-specific assembly of NMDA receptors in the ER, little is known. Some studies indicate that the C terminus of NR1 subunit has RXR motif, which can retain the NR1 subunit in the ER when expressed alone in heterogenous cells. And only when coexpressed with NR2 subunit, can this motif be masked. But the mechanism of the ER retention of NR2 subunit is still unclear. Other studies based on AMPA and KA receptors indicate that subunits making up NMDA receptors are organized into a dimer of dimers, but the domain involved in this process has not been determined. Some researches suggest that the amino terminal domain (ATD) is necessary for the functional assembly of NMDA receptors, but still others show different conclusion.Here we used a biophysical approach, fluorescence resonance energy transfer (FRET), 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. We also found that in the endoplasmic reticulum (ER), even the whole extracellular N-terminal domain (NT) was truncated, the NR subunit could form homomers or heteromers with each other in HEK293 cells, which indicate that the NT is not the crucial element for the receptor assembly. Furthermore, the analysis from live cell surface staining and whole cell patch clamp recording show that the amino-terminal domain (ATD)-deleted NR2A or NR2B subunits can form functional channels with the wild-type NR1a subunits. But the ATD-deleted NR1a subunits can only form functional channels with the wild-type NR2B but not NR2A subunit. More interestingly, when the ATD of NR2A is exchanged with that of NR2B, it can traffic to the cell membrane with both the wild-type and ATD-deleted NR1a subunits. When the ATD of NR2B is exchanged with that of NR2A subunit, it can form functional channels with only wild type NR1a subunit, but not ATD-deleted NR1a subunit. Taken together, our results suggest that the ATD is not important for the formation of functional NMDA receptors. But for NR1a/NR2A subtype, the ATD of NR2A may have the function of ER retention, and only with the help of NR1a ATD, can it form functional channels in the membrane.