Abstract
The genes encoding the ionotropic N-methyl-D-aspartate (NMDA) receptor subunits NR1a, NR2B and NR2D were cloned in the multi-copy yeast-Escherichia coli shuttle vectors pMBO1 and pMB02. The protease-deficient yeast Saccharomyces cerevisiae c13-ABYS-86 (leu-, ura-, his-) was transformed with the recombinant plasmids pMBNR1a (leu+), pMBNR1a/pMBNR2D (ura+), pMBNR1a/pMBNR2D/ pMBNR2B (his+) or pMBNR1a/pMBNR2A/pMBNR2B, respectively, and was used to express the different NMDA receptor subunit genes. Western-blotting analysis with the specific NMDA receptor antibodies showed a clear but differently strong expression of the recombinant receptor proteins which were found to be only partially glycosylated in the cell membranes of the recombinant yeast strains. By immunofluorescence microscopy using the specific subunit antibodies and fluorescence-labeled secondary antibodies, the distinctly expressed NR1a and NR2D subunits could be located in the plasma membrane of the transformed yeast cells. Pharmacological characterization of crude membrane preparations of the recombinant yeast cells expressing 1-3 NMDA receptor subunits showed saturable binding of the glycine antagonist [3H]MDL105,519 with different Kd values of 56.88+/-5.38 nM (NR1a), 1365.11+/-76 nM (NR1a/NR2D), 22.97+/-3.37 nM for NR1a/NR2B/NR2D and 7.4+/-1.2 nM for NR1a/NR2A/NR2B. The bound capacities were 13.07+/-0.92 (NRla), 14.63+/-0.50 (NR1a/NR2D), 12.85+/-1.68 (NR1a/NR2B/NR2D) and 8.3+/-0.7 (NR1a/NR2A/NR2B) pmol/mg membrane protein. The [3H]MDL105,519 binding was inhibited by the glycine antagonist 5,7-dichlorokynurenate (DCKA), ethyl-2-carboxy-4.6-dichloro-3-indoleacetate (ECDI) and itself, but not by glycine, D-serine and 1-amino-cyclopropanecarboxylic acid (ACPC). Each of these recombinant receptor proteins consisting both of NR1 and NR2 subunits also showed a specific binding site for the NMDA agonist glutamate when using L-[3H]glutamate as a radioligand. Analysis of saturation experiments revealed that this ligand binds to a specific site with Kd values of 536+/-43, 688+/-60, and 856+/-48 nM for NR1a/NR2B, NR1a/NR2D, and NR1a/NR2B/NR2D respectively.
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