Inducible expression of N-methyl-D-aspartate (NMDA) receptor channels from cloned cDNAs in CHO cells.
To develop a drug screening system, we introduced expression vectors carrying the mouse N-methyl-D-aspartate (NMDA) receptor channel epsilon1 and zeta1 subunit cDNAs under the promoter of the Drosophila heat shock protein hsp70 into Chinese hamster ovary (CHO) cells. We selected clonal cell lines by means of RNA blot hybridization and fura-2 fluorometry. One of these cell lines, ZE1-1, optimally expressed the epsilon1 and zeta1 subunit mRNAs when induced by an incubation at 43 degrees C for 2 h. Heated ZE1-1 cells exhibited the NMDA-induced intracellular Ca2+ elevation, whereas unheated they showed no such response. NMDA and L-glutamate, but not alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and kainate, induced an increase in the intracellular Ca2+ concentration. The response to the agonists was marginal in the absence of glycine, and diminished by Mg2+ and NMDA receptor antagonists. Furthermore, exposure to agonists of ZE1-1 cells expressing the epsilon1/zeta1 NMDA receptor channel resulted in the release of lactate dehydrogenase (LDH) activity in the culture medium indicating agonist-induced cell death. NMDA receptor antagonists inhibited the LDH activity release. These results suggest that ZE1-1 cells will provide a useful screening system for novel drugs acting on the epsilon1/zeta1 NMDA receptor channel.
Uchino
S
S
Yokohama Research Center, Mitsubishi Chemical Corporation, Kamoshida-cho, Aoba-ku, Japan.
Kudo
Y
Y
Watanabe
W
W
Nakajima-Iijima
S
S
Mishina
M
M
D000818:Animals; D016466:CHO Cells / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D051379:Mice; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism*
D018076:DNA, Complementary; D016194:Receptors, N-Methyl-D-Aspartate