The effect of increasing the expression of NMDA subunits in cerebellar granule cells (CGCs) by transfection was studied to determine how the availability of various NMDA subunits controls both the total pool of functional receptors and the synaptic pool. Overexpression of either NR2A or NR2B, but not splice variants of NR1, by transfection caused a significant increase in the total number of functional NMDA receptors and in surface NR1 subunit cluster density in CGCs in primary culture. These data solidify the central role of NR2 subunit availability in determining the number of cell surface receptors. Overexpression of either NR2A or NR2B significantly altered the deactivation kinetics of NMDA-mediated miniature EPSCs (NMDA-mEPSCs). However, there was no significant effect of NR2 subunit overexpression on the mEPSC amplitude or single-channel conductance. NR2 subunit overexpression did not change the rate of block by MK-801 of NMDA-mediated currents in excised patches from CGCs, indicating that subunit composition does not regulate peak open probability of the channel in CGCs. With the overexpression of a mutant of NR2B lacking the PDZ binding domain, there was an increase in the total number of NMDA receptors without a change in mEPSC kinetics. Therefore, the entry of NMDA receptors into the synapse requires a PDZ binding domain and is limited by means other than receptor subunit availability.
Functional N-methyl-D-aspartic acid (NMDA) receptors are formed from the assembly of NR1 and NR2 subunits. When expressed alone, the major NR1 splice variant and the NR2 subunits are retained in the endoplasmic reticulum (ER), reflecting a quality control mechanism found in many complex multisubunit proteins to ensure that only fully assembled and properly folded complexes reach the cell surface. Recent studies have identified an RRR motif in the C terminus of the NR1 subunit, which controls the ER retention of the unassembled subunit. Here we investigated the mechanisms controlling the ER retention of the NR2 subunit and the export of the assembled complex from the ER. We found that Tac chimeras of the C terminus of the NR2B subunit show that an ER retention signal is also present in the NR2B subunit. In assembled complexes, ER retention signals on the individual subunits must be overcome to allow the complex to leave the ER. One common mechanism involves mutual masking of the signals on the individual subunits. Our data do not support such a mechanism for regulating the release of assembled NMDA receptors from the ER. We found that the motif, HLFY, immediately following transmembrane domain 4 of the NR2 subunit, is required for the assembled complex to exit from the ER. Mutation of this motif allowed the assembly of NR1 and NR2 subunits into a complex that was functional, based on MK-801 binding, but it is retained in the ER. These results are consistent with HLFY functioning as a signal that is necessary for the release of the assembled functional NMDA receptor complex from the ER. N-Methyl-D-aspartic acid (NMDA)1 receptors are multisubunit complexes assembled from three subunit classes, NR1, NR2, and NR3. Co-assembly of NR1 and NR2 subunits is required to form a functional channel, generally considered to be a tetramer containing two NR1 and two NR2 subunits (1).NR3 subunits can assemble with NR1 alone to form a glycine receptor (2), and they also can have a modulatory role on NR1/NR2 complexes by associating in a yet undefined fashion (3). NR1 is a single subunit that can be expressed in eight splice variants, which have distinct trafficking and functional properties. There are four NR2 subunits, NR2A-D, each of which differs in its distribution and developmental expressions. The type of NR2 subunit present in the complex has a major impact on the functional properties of the channel. For example, NR1/ NR2A channels display rapidly desensitizing currents and close association with the synapse compared with NR1/NR2B channels (4, 5). Therefore, the subunit composition of NMDA receptors is a major determinant of NMDA receptor-mediated activity and localization in the central nervous system. Several splice variants of NR1 and different subunits of NR2 are present in the same neurons, leading to multiple receptors with different functional and trafficking properties (6). For example, a neuron that expresses NR2A and NR2B subunits can contain three functionally distinct receptors: NR1/NR2A, NR1/NR2B, and NR1/NR2A...
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