The Mechanisms Of C Terminus Of NR2B Subunits Regulate On NMDA Receptor Trafficking And Surface Expression

The N-methyl-d-aspartate receptor(NMDAR)is one of the glutamate-gated ion channels,and plays a central role in excitatory synapses where it has been implicated in multiple functions associated with synaptic plasticity.While this receptor has been intensively studied with respect to its physiology and pharmacology,its cell-biological properties,such as subunit assembly,post-translational processing and trafficking in neurons,are only beginning to be addressed.The NR1 subunit is a gene family,which can generate eight NR1 splice variants by alternative splicing.The NR2 genes encode four subunits named NR2A,NR2B,NR2C and NR2D.The NR3 subunit has two genes,which are NR3A and NR.3B.Functional NMDARs are made up of NR1 and NR2 subunits,forming a tetrameric complex.It is widely recognized that the NR1 subunit is essential for the functional NMDA receptor channels,while various combinations of NR2 and NR1 subunits could endow NMDA receptor channels with different functional properties.Neither NR1 nor NR2 subunits form functional receptors when expressed alone in heterologous cells.Early studies showed that when expressed alone,these subunits are retained in the endoplasmic reticulum(ER).Subsequent studies,however,showed that only the NRI-1 splice variant,the major splice variant found in brain,is ER retained.Two distinct motifs in the C-terminal domain control the ER retention of the NR1 subunit.The RRR motif in the C1 splice cassette is an ER retention signal,and STVV,the PDZ-interacting domain of the C2' cassette,serves as an ER exit signal.Otherwise,activation of PKC also promotes the NRI-la to deliver to the cell membrane.However,the mechanism of ER retention of NR2 subunit is still unknown.Our previous studies did not find any specific domain of NR2B C-terminus which can determine the ER retention of NR2B subunit.It implies that the ER retention of NR2B subunit is more complex than that of NR1 subunit.To address the role of NR2B C-terminus in ER retention of NR2B subunit,we generated a NR2B_NR4aCchimera that is the NR2B C-terminus replaces with the C terminus of NR1-4a.Interestingly,expression of this chimera can reach to the cell membrane alone in HEK293 cells.It suggests that the C terminus of NR2B subunit is essential for the surface expression of NR2B subunit by itself.In addition,it is a mystery whether the NR2B subunit can form a functional homomeric channel in vivo.Since the NR2B_NR4acchimera can express on the cell surface,we try to record the recombinant NMDAR currents by whole-cell patch clamp in cells transfected with this chimera.Unfortunately,the NR2B_NR4aCchimera can not be evoked any currents by applying a focal saturating dose of glutamate and glycine. This result suggests the NR2B subunit can not form a functional channel by itself.Secondly,the cytoplasmic C-terminal domains of NR2 subunits have been proposed to modulate the assembly and trafficking of NMDA receptors.However, questions remain concerning which domains in the C-terminus of NR2 subunits control the assembly of receptor complexes,and how the assembled complexes are selectively trafficked through the various cellular compartments such as ER to the cell surface.In the present study,we find that the three amino-acid tail after the TM4 region of NR2B subunit is necessary for surface expression of functional NMDA receptors,while truncation with only two amino-acids following the TM4 region (2Bâ–³2)completely eliminates surface expression of the NMDA receptor on coexpression with NR1-1a in HEK293 cells.FRET(fluorescence resonance energy transfer)analysis showes that the 2Bâ–³2 truncations are able to form homomers and heteromers on co-expression with NR1-1a.Furthermore,when NR2â–³2 subunits are cotransfected with either the NR1-4a or NR1-1aAAA mutant,functional NMDA receptors are detected in the transfected HEK293 cells.Futhermore,we find that the replacement of five residues after TM4 with alanines give results indistinguishable from those of 2BA5(EHLFY),demonstrating the short tail following the TM4 of NR2B subunit is not sequence specificity-dependent.Taken together,our results show that the C-terminus of the NR2B subunits is not necessary for the assembly of NMDA receptor complexes,whereas a three amino acid long cytoplasmic tail following the TM4 of NR.2B subunits is sufficient to overcome the ER retention existing in the C-terminus of NR1,allowing the assembled NMDA receptors to reach the cell surface.Finaly,we transfected these C terminal truncations of NR2A and NR2B subunits into the cultured hippocampal neuron respectively,and detected the surface staining of these truncations.Our results show that a small number of surface expressed NMDARs containing 2Aâ–³2 was detected at DIV7 in cultured hippocampal neurons, while no 2BA2 containing NMDARs were detected on the cell surface.When co-transfected the NR1-4a with 2BA2 into neurons,however,the 2BA2 can be delivered to the membrane surface.As we known,there are eight splice variants of NR1 subunit in neurons.If the NR2 subunit can randomly assemble with one of the NR1 splicing variants,it is impossible that the 2BA2 is retained in the ER.So our results suggest one likely reason,which is the endogenous NR1 splice variants may have different preferences for assembling with NR2A versus NP,2B subunit in cultured hippocampal neurons.