Distribution Pattern Of NMDA Receptor Subtypes Mediated By NR2 Subunit C Terminus And Interaction With PSD-MAGUK Family Proteins

N-methyl-D-aspartate(NMDA)receptors,one of three main classes of ionotropic glutamate receptor,play major roles in synaptic plasticity, synaptogenesis and excitotoxicity.Functional NMDA receptors are believed to be hetero-tetrameric complexes assembled from two NR1 and two NR2 subunits.The NR1 subunits have eight splice variants and are obligatory for the formation of functional NMDA receptor channels, while the NR2 subunits,containing four different genes NR2A-2D.The subunit composition defines many properties of NMDA receptors essential for synaptic plasticity.Particularly crucial are the NR2 subunits, which set the voltage dependence of the pore Mg²⁺block,the time course of NMDA receptor currents,and the intracellular binding partners important for signal transduction.The membrane distribution pattern and synaptic localization of NMDA receptors,which are important for the regulation of synaptic strength and the determination of the direction of synaptic plasticity,also occur in a NR2-dependent manner.Previous studies have suggested that in mature neurons NR2A-containing NMDA receptors,unlike those containing NR2B,are located almost exclusively within synapse,although there are evidences suggested that NR2A-containing NMDA receptors can also located in extrasynaptic compartments.However,the observation has been report that before formation of synapses surface NMDA receptors transport along the dendrite in a clustering manner.Thus,the receptors clustering outside the sites of excitatory synaptic contact may not be detected by electrophysiological application.Comprehensive morphological analysis and comparisons of the membrane distribution patterns of NR2A-and NR2B-containing NMDA receptors in neuron are still required.In the present study we generated Cy3-conjugated fab fragment of anti-GFP antibody to direct label surface expressed GFP-NR2A or GFP-NR2B protein.As NR2 subunit is retained in the endoplasmic reticulum in the absence of coassembly with NR1,we demonstrated the membrane distribution of NR2A- and NR2B-containing NMDA receptors in cultured hippocampal neuron by surface NR2 staining.Our study indicated that the surface distribution of NR2A-containing NMDARs prefered to be more clustered than that of NR2B-containing ones.By exchanging the carboxyl termini of NR2A and NR2B subunit, surface NR2B-containing NMDA receptors occurred more in a clustering manner,whereas NR2A-containing ones more diffusedly.However,PDZ binding failed to be responsible for the clustering of surface NR2A-containing receptors.We then found that in mature neuron PSD-95 clusters more specifically associated with the surface clusters of NR2A-containing NMDA receptors,whereas SAP-102 clusters similarly associated with those of NR2A-and NR2B-containing NMDA receptors. The PDZ binding never contributed to this specific association.In addition,during early development of neuron,expression of NR2A subunit promoted the clustering of PSD-95.Therefore,our results suggested that the differential membrane distribution pattern of NR2A-and NR2B-containing NMDARs may be determined by the carboxyl termini of NR2 subunits but not completely depend on the PDZ binding, and the specific distribution pattern of NR2A-containing NMDA receptors may be related to the specific association with PSD-95.