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Silencing of ZnT-1 expression enhances heavy metal influx and toxicity

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Abstract

ZnT-1 reduces intracellular zinc accumulation and confers resistance against cadmium toxicity by a mechanism which is still unresolved. A functional link between the L-type calcium channels (LTCC) and ZnT-1 has been suggested, indicating that ZnT-1 may regulate ion permeation through this pathway. In the present study, immunohistochemical analysis revealed a striking overlap of the expression pattern of LTCC and ZnT-1 in cardiac tissue and brain. Using siRNA to silence ZnT-1 expression, we then assessed the role of ZnT-1 in regulating cation permeation through the L-type Ca2+ channels in cells that are vulnerable to heavy metal permeation. Transfection of cortical neurons with ZnT-1 siRNA resulted in about 70% reduction of ZnT-1 expression and increased Ca2+ influx via LTCC by approximately fourfold. Moreover, ZnT-1 siRNA transfected neurons showed ∼30% increase in synaptic release, monitored using the FM1-43 dye. An increased cation influx rate, through the LTCC, was also recorded for Zn2+ and Cd2+ in cells treated with the ZnT-1 siRNA. Furthermore, Cd2+-induced neuronal death increased by approximately twofold after transfection with ZnT-1 siRNA. In addition, ZnT-1 siRNA transfection of the ovarian granulosa cell line, POGRS1, resulted in a twofold increase in Cd2+ influx rate via the LTCC. Finally, a robust nimodipine-sensitive Cd2+ influx was observed using a low extracellular Cd2+ concentration (5 μM) in neurons and testicular slice cultures, attesting to the relevance of the LTCC pathway to heavy metal toxicity. Taken together, our results indicate that endogenously-expressed ZnT-1, by modulating LTCC, has a dual role: regulating calcium influx, and attenuating Cd2+ and Zn2+ permeation and toxicity in neurons and other cell types.

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Abbreviations

LTCC:

L-type calcium channels

siRNA:

small interfering RNA

MT:

Metallothionein

TPEN,N,N,N′,N′:

Tetrakis(2-pyrifylmethyl)ethyllenediamine

SDS:

Sodium dodecyl sulfate

LDH:

Lactate dehydrogenase

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Acknowledgements

We thank the fellowship generously donated by Daniel Falkner to E.O. This work was partially supported by the ISF (to I.S. and to W.F.S.) and the BSF (to M.H.) and by the BG Negev (to I.S. and M.H). We also thank Dr. Daniel Gitler for his invaluable advice on the analysis of the synaptic transmission data.

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Authors and Affiliations

  1. Department of Physiology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Ehud Ohana & Israel Sekler

  2. Department of Morphology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Tehila Kaisman, Nicol Kahn, William F. Silverman & Michal Hershfinkel

  3. Zlotowski Center for Neuroscience, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Ehud Ohana, Israel Sekler, Tehila Kaisman, Nicol Kahn, Joshua Cove, William F. Silverman & Michal Hershfinkel

  4. Department of Life Sciences, Faculty of Natural Sciences, Ben Gurion University of the Negev, Beer-Sheva, 84105, Israel

    Joshua Cove

  5. Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel

    Abraham Amsterdam

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  1. Ehud Ohana
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  2. Israel Sekler
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  3. Tehila Kaisman
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  8. Michal Hershfinkel
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Correspondence to Michal Hershfinkel.

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Ohana, E., Sekler, I., Kaisman, T. et al. Silencing of ZnT-1 expression enhances heavy metal influx and toxicity. J Mol Med 84, 753–763 (2006). https://doi.org/10.1007/s00109-006-0062-4

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