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Abstract

Data from both in vivo and in vitro studies indicate that prolonged exposure of neurons to glutamate results in over-activation of N-methyl-D-aspartate (NMDA)-type receptors, allowing an increase in [Ca²⁺]i and resulting in cell death. However, details of the molecular mechanisms, particularly the role of the NMDA receptor's structure, function, and subunit composition that underlie these events have been difficult to study in neuronal systems. We have co-expressed recombinant NMDA receptors with the marker protein (ß-galactosidase in human embryonic kidney cells and determined the amount of viable transfected cells by immunostaining with NR1 specific antibodies and / or assaying for ß-gal expression. Incubation of NR1 / NR2A/ ß-gal transfected HEK-293 cells in bicarbonate-buffered physiological salt solution for 6 hours resulted in loss of transfected cells when NMDA was included. This effect was dose-dependent with an EC₅₀ between 150-300 | μM NMDA. When these experiments were repeated with cells expressing Ca⁺²-impermeable mutant NR1 (N598R) / NR2A receptors, cell death was diminished by -75%. On the other hand, in experiments with wild-type NR1/NR2Atransfected cells, substitution of N-methyl-glucamine for Na⁺ in the salt solution decreased cell death by 50%, suggesting that cytotoxicity was mediated in part by Na⁺ flux through NMDA receptors. Using this system, it was shown that the rapid desensitization of non-NMDA receptors is protective against agonist-induced cell death. In addition, the effect of zinc on NMDA toxicity was examined. In NR1 / NR2A-transfected cells, zinc reduced the cyotoxicity of NMDA with an IC₅₀ of -500 nM. Furthermore, it was shown that zinc inhibits the peak glutamate-evoked current responses and accelerates desensitization in whole-cell patch clamp recordings from NR1 / NR2A and NR1 / NR2B transfected cells. However, NR1 / NR2A was ~20-fold more sensitive to zinc inhibition than NR1 / NR2B, with IC₅₀S of~500 nM and -10 μM, respectively. Finally, the effect of serum albumin on NMDA toxicity was analyzed. It was shown that serum albumin does not potentiate NMDA toxicity in transfected HEK-293 cells. However, the inhibitory effect of zinc on NMDA toxicity was reduced in the presence of serum albumin. Hence, increased concentrations of serum albumin in the brain observed during pathological conditions, could disrupt the regulatory function of Zn²⁺ on NMDA receptors.