A Study Of Function Of GluN2A Subunit ATD Domain In Regulatiog NMDA Receptor Assembly，Intracellular Trafficking And Activity Dependent Surface Expression

N-methyl-D-aspartate (NMDA) receptor is an important iontropic glutamate receptor in central nervous system, and it is widely expressed in the central nervous system. NMDA receptor has unique and complex function. For example, NMDA receptor is ligand and voltage double gated receptor, and it is highly permeable to Ca2+. Besides, NMDA receptor is coupled to multiple signaling pathways in neurons. All of these functional properties promised that NMDA receptor play vital roles in excitatory synaptic transmission and neuron plasticity. As a result, the proper function of NMDA receptor is the prerequisite for learning and memory. On the other hand, NMDA receptor is also involved in many neuronal degeneration diseases. Therefore, to clarify the function of NMDA receptor is of great significance on understanding the mechanisms of neuronal information processing, higher brain function and neuropsychiatric diseases.NMDA receptor is a tetramer and usually composed of two obligatory GluNl subunits and two modulatory GluN2or GluN3subunits. Modulatory subunits are the foundation of composition and function diversity of NMDA receptor, and in which GluN2A and GluN2B have attracted major attention because they participate in many important brain functions. Although they have similar structure, the function of GluN2A and GluN2B containing NMDA receptors are highly different, they even play controversial roles in neuron plasticity and neuron survival. Obviously, neurons adopt a dedicated mechanism to separately regulate the expression of GluN2A and GluN2B containing NMDA receptors. However, it has not been clarified yet.In this work, we identified an new intracellular mechanism to modulate the neuron surface ratio of GluN2A/GluN2B. We identified a subunit specific Endoplasmic Reticulum (ER) retention signal located in the ATD domain of GluN2A subunit, which is absent from the homologous region of GluN2B. This property retained a number of unassembled GluN2A homodimers in dendrite ER. Under neuronal activities, when GluN2A containing NMDA receptors are required, the GluN2A homodimers that were stored in ER would be released and complete assembly and express on the neuron membrane and to change the surface ratio of GluN2A/GluN2B. Further we screened the involved ER chaperone proteins by proteomic approach and found that Bip has a strong interaction with GluN2A rather than other NMDA receptor subunits. As an ER protein, Bip mediated the GluN2A/GluN1assembly under specific neuronal activity. Once the interaction of Bip and GluN2A was interrupted by peptides, GluN2A can no longer response to the neuronal activities. Thus we identified a previously unknown mechanism to store and modulate multiple-subunit neuronal receptors in dendrite ER.Besides, in this work we also studied the function of GluN2A ATD in NMDA receptor assembly. We found that the ATD domain of GluN2A can form homodimer when it was expressed in heterogeneous system as transmembrane domain and the homodimer formation depends on inter-subunit covalent bond of two cysteine residues. Nevertheless it is required for GluN2A dimerization but not NMDA receptor assembly. This result suggested that NMDA receptor assembly is not as AMPA receptor, starting at the ATD domain dimerization, but more likely to be determined by the transmembrane region. It suggested that GluN2A and GluN1play unequal roles in NMDA receptor assembly.In summary, in this work combining molecular biology and cell biology, we illustrated function of GluN2A subunit ATD domain in regulating subunit-selective expression of NMDA receptors. And for the first time, we proved the new mechanism that dendrite ER is responsible for assembly, storage and synaptic activity dependent release of multiple-subunits receptors. These findings are of significant importance for further elucidating the function of NMDA receptor in neuronal plasticity, as well as neuronal death and survival.