| 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 NR3 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.NR1 subunit is widespread in the nervous system, whereas the expression of NR2A-2D subunits, with distinct developmental and regional patterns, is generally more restricted than the homogeneous distribution of NR1 subunit. The predominantly expressed NR2 subunits in hippocampus and neocortex are NR2A and NR2B, They have different developmental expression profiles - there is a developmental decrease in expression of the NR2B subunit and an increase in expression of the NR2A subunit, which endow the NMDA receptor with distinct channel properties. A change in functional properties of the NMDA receptors has been invoked as a potential mechanism contributing to the loss of synaptic plasticity during brain maturation. From this, it is important to well understand the structural and functional diversity of NMDA receptors in relation to NMDA receptor subunit composition and spatial distribution at excitatory synaptic sites during development.In the present years, a great body of the evidence demonstrates that the C terminus of AMPA receptors play a key role in their endocytosis and insertion, especially the AMPA receptors will have endocytosis or insert into the synapes through interacting with the other specific protein or phosphorylation. Resent studies demonstrate that, such as GABAAR1 subunit of GABA receptor, KA2 and GluR6 subunit of KA receptor, there are some functional regions in that of C terminus which can regulate these subunits targeting to the cell membrane surface. While little was known whether NMDA receptor's NR2B subunit has the similar domains. The NR2B subunit have a long cytoplasmic tail(644aa), so it predicts that there may be some functional domains in this tail.On the other hand, our previous studies have shown that the NR2B construct with a GFP tag in its N-terminus cotransfected with NR1 subunit can reach the cell surface and be detected by surface staining with anti-GFP antibody in live cells. The NR2B construct will be retained in the endoplasmic reticulum (ER) when expressed alone in heterologous cells.In our study,C terminus of the GFP tagged NR2B with various deficient mutants (GFP-NR2BA1-A14) were constructed and verified. When expressing these mutants ino HEK 293 cell, we can observe the well diffuse fluorescence in the transfected cell .By using live surface staining and electrophysiology, research was made on following aspects:(1) To identify whether C terminus of NR2B subunit has some functional domains that can regulate NR2B subunit trafficking to the cell membrane, GFP-NR2B A1-A14 constructs were transfected into HEK 293 cells, and after 48 hours were incubated with rabbit polyclonal anti-GFP antibody, then...
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