The Function Of The N Terminal Domain In Assembly Of N-methyl-d-aspartate Receptors Analyzed By NR1-GluR2 Subunit Chimeras

A central assumption in neurobiology holds that synapse formation and synaptic plasticity underlie the creation of functional neural circuits and nervous system plasticity. Two major subtypes of ionotropic glutamate receptors(GluRs), N-methyl-D-aspartate (NMDA)-type and a-amino-3-hydroxy-5-methyl-4- isoxazole-propionate(AMPA)-type receptors, are found localized on glutamatergic postsynaptic excitatory synapse. They fulfill coordinately the complex functional roles of glutamatergic neurotransmission. Accumulating evidence indicates that NMDA receptors are involved in many complex physiological and neuropathological mechanisms, such as neuronal developmental plasticity, long-term potentiation (LTP), learning and memory, excitotoxicity and neurodegenerative diseases etc.Three gene families that encode NMDA receptor subunits have been identified: NR1, NR2A-D, and NR3A,3B subunit. Alternative splicing generates eight isoforms for NR1 subfamily. The NR2 genes encode of four subunits named NR2A, NR2B, NR2C and NR2D. It is widely recognized that the NR1 subunit is essential to the functional NMDA receptor channels, while various combinations of NR2 and NR1 subunits could endow NMDA receptor channel with different functional properties. Meanwhile, the splice variants of NR1 subunit could also produce differences in the properties of NMDA receptor channel.NMDA receptors are thought to be tetrameric complex mainly composed of NR1 and NR2 subunits.However, the pairing rules governing the selectivity of theassembly of NMDA receptors are still far from being well understood. And regions on the NMDA receptors subunits mediating assembly of homomeric or heteromeric receptors have not been identified so far. Previous biochemical studies indicate that the N-termini of the subunits are major determinants for NMDA receptor assembling. In the present years, the research on receptor assembly depend on the biochemistry method and technology ,such as the co-immunoprecipitation. However, experiment is very specific and could repeat well , some result would be not true since the experiment steps and without the physiology environment. Recently the fluorescence resonance energy transfer(FRET) is a specific way ,which could detect two molecular distance less than 1-10 nm.It could explain the problem that moleculars interaction and formation changing. And the resolution is beyond the normal microscope.In order to explain the N terminal function for the NMDA receptor assembly.The NR1 subunit become our first choice. Not only due to the difference NR1 subtype could lead to the different function for the NMDA receptor, but also the NR1 subuint could saparete almost in the all the brain. N- terminus of the YFP and CFP tagged NR1 with various deficient mutants were constructed and verified. When expressing these mutants into HEK 293 cell, we can observe the well diffuse fluorescence in the transfected cell. By using live surface staining and living cell fluorescence resonance energy transfer, research was made on following aspects:(1) To identify whether N terminus of NR1a subunit has some functional domains that can regulate NR1a and NR2B complex trafficking to the cell membrane, XFP-NR1-ATD-GluR2 , XFP-NR1-S1-GluR2 ,XFP-NR1-NT-GluR2. constructs were transfected into HEK 293 cells, and after 48 hours were incubated with rabbit polyclonal anti-GFP antibody, then incubated with Cy3-conjugated goat anti-rabbit antibody. On the one hand, the NR1 Subunit chimeras have different surface expression with the wide type. On the other hand , the HEK293 cells which was transfacted by NR1 subunit chimeras and the NR2B have no NMDAR current rather than the wide type. Therefore, our results suggest that the N terminus of NR1a subunit has important role on the NR1a and NR2B complex surface expression of itself.(2) In order to find out whether the CFP-NR1a-NT-GluR2, YFP-NR1-NT-GluR2 could form the homopolymer, we transfacted these into the HEK293 and using three-cube FRET measurement detected the FRET. Then we find that these subunit could form the homopolymer in cell. We find the similar results from the CFP-NR1a-ATD-GluR2 , YFP-NR1a-ATD-GluR2 , CFP-NR1a-S1-GluR2 , YFP-NR1a-S1-GluR2 respectively cotransfacted into the HEK293 cell. It infer that the chimeras of NR1a is no effect of the whole subunit topograph. They could assembly easily and correctly through interaction between NR1and GluR2. On the other hand we found that the CFP-NR1-NT-GluR2 could assembly easily with the NR1 and NR2B subunit. It might infer that the chimeras construction is no mean to the subunit topograph and we could find the N terminal is not essential for the NMDA subunit assembly.