EXPRESSION OF N-METHYL-D-ASPARTATE RECEPTORS USING VACCINIA VIRUS CAUSES EXCITOTOXIC DEATH IN HUMAN KIDNEY-CELLS

N-Methyl-D-Aspartate (NMDA) receptors containing NR1 and NR2A subunits have been expressed with high efficiency in Human Embryonic Kidney 29 3 cells with the aid of a recombinant vaccinia virus. This expression system produced functional receptors that sustained calcium influxes d ependent on receptor agonists and inhibited by receptor antagonists. I mmunocytochemistry of the recombinant receptors demonstrated that they were properly arranged in membrane structures. The entrance of calciu m through the recombinant receptors induced delayed toxicity, demonstr ated by approximately a three-fold increase in the number of dead cell s obtained 12 h after the antagonist 2-amino-phosphopentanoic acid (DL -AP5) was removed from the culture. This result correlated with more t han 88% inhibition in the expression of a reporter gene 24 h after ant agonist removal. Calcium toxicity was completely abolished by specific antagonists of the NMDA receptor. Treatment of cell extracts with N-g lycosydase showed that both receptor subunits were N-glycosylated. Tun icamycin prevented calcium toxicity; gel electrophoresis studies showe d that this protection was likely due to degradation of the NR1 subuni t.). Cell. Biochem. 72:135-144, 1999. (C) 1999 Wiley-Liss, Inc.