L-type Ca 2+ channel-mediated Zn 2+ toxicity and modulation by ZnT-1 in PC12 cells

Abstract

In view of evidence that Zn 2+ neurotoxicity contributes to some forms of pathological neuronal death, we developed a model of Zn 2+ neurotoxicity in a cell line amenable to genetic manipulations. Exposure to 500 μM ZnCl 2 for 15 min under depolarizing conditions resulted in modest levels of PC12 cell death, that was reduced by the L-type Ca 2+ channel antagonist, nimodipine, and increased by the L-type Ca 2+ channel opener, S(−)-Bay K 8644. At lower insult levels (200 μM Zn 2++Bay K 8644), Zn 2+-induced death appeared apoptotic under electron microscopy and was sensitive to the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-CH 2F (Z-VAD); at higher insult levels (1000 μM+Bay K 8644), cells underwent necrosis insensitive to Z-VAD. To test the hypothesis that the plasma membrane transporter, ZnT-1, modulates Zn 2+ neurotoxicity, we generated stable PC12 cell lines overexpressing wild type or dominant negative forms of rat ZnT-1 (rZnT-1). Clones T9 and T23 overexpressing wild type rZnT-1 exhibited enhanced Zn 2+ efflux and reduced vulnerability to Zn 2+-induced death compared to the parental line, whereas clones D5 and D16 expressing dominant negative rZnT-1 exhibited the opposite characteristics.