Becker J, Li Z, Noe C R
Institut für Pharmazeutische Chemie, Frankfurt, Germany.
Eur J Biochem. 1998 Sep 1;256(2):427-35. doi: 10.1046/j.1432-1327.1998.2560427.x.
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.
编码离子型N-甲基-D-天冬氨酸（NMDA）受体亚基NR1a、NR2B和NR2D的基因被克隆到多拷贝酵母-大肠杆菌穿梭载体pMBO1和pMB02中。用重组质粒pMBNR1a（亮氨酸+）、pMBNR1a/pMBNR2D（尿嘧啶+）、pMBNR1a/pMBNR2D/pMBNR2B（组氨酸+）或pMBNR1a/pMBNR2A/pMBNR2B分别转化蛋白酶缺陷型酵母酿酒酵母c13-ABYS-86（亮氨酸-、尿嘧啶-、组氨酸-），并用于表达不同的NMDA受体亚基基因。用特异性NMDA受体抗体进行的蛋白质免疫印迹分析显示，重组受体蛋白有明显但强度不同的表达，这些蛋白在重组酵母菌株的细胞膜中仅部分糖基化。通过使用特异性亚基抗体和荧光标记二抗的免疫荧光显微镜观察，明显表达的NR1a和NR2D亚基可定位在转化酵母细胞的质膜中。对表达1 - 3个NMDA受体亚基的重组酵母细胞的粗制膜制剂进行药理学特性分析，结果显示甘氨酸拮抗剂[3H]MDL105,519有饱和结合，不同解离常数（Kd）值分别为56.88±5.38 nM（NR1a）、1365.11±76 nM（NR1a/NR2D）、22.97±3.37 nM（NR1a/NR2B/NR2D）和7.4±1.2 nM（NR1a/NR2A/NR2B）。结合容量分别为13.07±0.92（NR1a）、14.63±0.50（NR1a/NR2D）、12.85±1.68（NR1a/NR2B/NR2D）和8.3±0.7（NR1a/NR2A/NR2B）pmol/mg膜蛋白。[3H]MDL105,519的结合被甘氨酸拮抗剂5,7 - 二氯犬尿氨酸（DCKA）、乙基 - 2 - 羧基 - 4,6 - 二氯 - 3 - 吲哚乙酸（ECDI）及其本身抑制，但不被甘氨酸、D - 丝氨酸和1 - 氨基环丙烷羧酸（ACPC）抑制。当使用L - [3H]谷氨酸作为放射性配体时，这些由NR1和NR2亚基组成的重组受体蛋白中的每一种也显示出对NMDA激动剂谷氨酸的特异性结合位点。饱和实验分析表明，该配体分别以536±43、688±60和856±48 nM的Kd值与NR1a/NR2B、NR1a/NR2D和NR1a/NR2B/NR2D的特定位点结合。
