Expression of N-methyl-D-aspartate receptors using vaccinia virus causes excitotoxic death in human kidney cells

Abstract

N-Methyl-D-Aspartate (NMDA) receptors containing NR1 and NR2A subunits have been expressed with high efficiency in Human Embryonic Kidney 293 cells with the aid of a recombinant vaccinia virus. This expression system produced functional receptors that sustained calcium influxes dependent on receptor agonists and inhibited by receptor antagonists. Immunocytochemistry of the recombinant receptors demonstrated that they were properly arranged in membrane structures. The entrance of calcium through the recombinant receptors induced delayed toxicity, demonstrated by approximately a three- fold increase in the number of dead cells obtained 12 h after the antagonist 2-amino-phosphopentanoic acid (DL-AP5) was removed from the culture. This result correlated with more than 88% inhibition in the expression of a reporter gene 24 h after antagonist removal. Calcium toxicity was completely abolished by specific antagonists of the NMDA receptor. Treatment of cell extracts with N-glycosydase showed that both receptor subunits were N- glycosylated. Tunicamycin prevented calcium toxicity; gel electrophoresis studies showed that this protection was likely due to degradation of the NR1 subunit.