Skip to main content
Log in

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

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

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

References

  1. Palmiter RD, Findley SD (1995) Cloning and functional characterization of a mammalian zinc transporter that confers resistance to zinc. EMBO-J 14:639–649

    PubMed  CAS  Google Scholar 

  2. Nolte C, Gore A, Sekler I, Kresse W, Hershfinkel M, Hoffmann A, Kettenmann H, Moran A (2004) ZnT-1 expression in astroglial cells protects against zinc toxicity and slows the accumulation of intracellular zinc. Glia 48:145–155

    Article  PubMed  Google Scholar 

  3. Segal D, Ohana E, Besser L, Hershfinkel M, Moran A, Sekler I (2004) A role for ZnT-1 in regulating cellular cation influx. Biochem Biophys Res Commun 323:1145–1150

    Article  PubMed  CAS  Google Scholar 

  4. Atar D, Backx PH, Appel MM, Gao WD, Marban E (1995) Excitation–transcription coupling mediated by zinc influx through voltage-dependent calcium channels. J Biol Chem 270:2473–2477

    Article  PubMed  CAS  Google Scholar 

  5. Sensi SL, Canzoniero LM, Yu SP, Ying HS, Koh JY, Kerchner GA, Choi DW (1997) Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17:9554–9564

    PubMed  CAS  Google Scholar 

  6. Usai C, Barberis A, Moccagatta L, Marchetti C (1999) Pathways of cadmium influx in mammalian neurons. J Neurochem 72:2154–2161

    Article  PubMed  CAS  Google Scholar 

  7. Choi DW (1996) Zinc neurotoxicity may contribute to selective neuronal death following transient global cerebral ischemia. Cold Spring Harbor Symp Quant Biol 61:385–387

    PubMed  CAS  Google Scholar 

  8. Cherny RA, Barnham KJ, Lynch T, Volitakis I, Li QX, McLean CA, Multhaup G, Beyreuther K, Tanzi RE, Masters CL, Bush AI (2000) Chelation and intercalation: complementary properties in a compound for the treatment of Alzheimer’s disease. J Struct Biol 130:209–216

    Article  PubMed  CAS  Google Scholar 

  9. Fern R, Black JA, Ransom BR, Waxman SG (1996) Cd(2+)-induced injury in CNS white matter. J Neurophysiol 76:3264–3273

    PubMed  CAS  Google Scholar 

  10. Benoff S, Jacob A, Hurley IR (2000) Male infertility and environmental exposure to lead and cadmium. Hum Reprod Update 6:107–121

    Article  PubMed  CAS  Google Scholar 

  11. Pant N, Upadhyay G, Pandey S, Mathur N, Saxena DK, Srivastava SP, Xu LC, Wang SY, Yang XF, Wang XR (2003) Lead and cadmium concentration in the seminal plasma of men in the general population: correlation with sperm quality. Reprod Toxicol 17:447–450

    Article  PubMed  CAS  Google Scholar 

  12. Johnson MD, Kenney N, Stoica A, Hilakivi-Clarke L, Singh B, Chepko G, Clarke R, Sholler PF, Lirio AA, Foss C, Reiter R, Trock B, Paik S, Martin MB (2003) Cadmium mimics the in vivo effects of estrogen in the uterus and mammary gland. Nat Med 9:1081–1084

    Article  PubMed  CAS  Google Scholar 

  13. Hasumi M, Suzuki K, Matsui H, Koike H, Ito K, Yamanaka H (2003) Regulation of metallothionein and zinc transporter expression in human prostate cancer cells and tissues. Cancer Lett 200:187–195

    Article  PubMed  CAS  Google Scholar 

  14. Andrews GK (2001) Cellular zinc sensors: MTF-1 regulation of gene expression. BioMetals 14:223–237

    Article  PubMed  CAS  Google Scholar 

  15. Takiguchi M, Cherrington NJ, Hartley DP, Klaassen CD, Waalkes MP (2001) Cyproterone acetate induces a cellular tolerance to cadmium in rat liver epithelial cells involving reduced cadmium accumulation. Toxicology 165:13–25

    Article  PubMed  CAS  Google Scholar 

  16. Trebak M, Vazquez G, Bird GS, Putney JW Jr (2003) The TRPC3/6/7 subfamily of cation channels. Cell Calcium 33:451–461

    Article  PubMed  CAS  Google Scholar 

  17. Andrews GK, Wang H, Dey SK, Palmiter RD (2004) Mouse zinc transporter 1 gene provides an essential function during early embryonic development. Genesis 40:74–81

    Article  PubMed  CAS  Google Scholar 

  18. Moss EG, Taylor JM (2003) Small-interfering RNAs in the radar of the interferon system. Nat Cell Biol 5:771–772

    Article  PubMed  CAS  Google Scholar 

  19. Ohana E, Segal D, Palty R, Ton-That D, Moran A, Sensi SL, Weiss JH, Hershfinkel M, Sekler I (2004) A sodium zinc exchange mechanism is mediating extrusion of zinc in mammalian cells. J Biol Chem 279:4278–4284

    Article  PubMed  CAS  Google Scholar 

  20. Selvaraj N, Israeli D, Amsterdam A (1996) Partial sequencing of the rat steroidogenic acute regulatory protein message from immortalized granulosa cells: regulation by gonadotropins and isoproterenol. Mol Cell Endocrinol 123:171–177

    Article  PubMed  CAS  Google Scholar 

  21. Nitzan YB, Sekler I, Hershfinkel M, Moran A, Silverman WF (2002) Postnatal regulation of ZnT-1 expression in the mouse brain. Brain Res Dev Brain Res 137:149–157

    Article  PubMed  CAS  Google Scholar 

  22. Sekler I, Moran A, Hershfinkel M, Dori A, Margulis A, Birenzweig N, Nitzan Y, Silverman WF (2002) Distribution of the zinc transporter ZnT-1 in comparison with chelatable zinc in the mouse brain. J Comp Neurol 447:201–209

    Article  PubMed  CAS  Google Scholar 

  23. Hershfinkel M, Moran A, Grossman N, Sekler I (2001) A zinc-sensing receptor triggers the release of intracellular Ca2+ and regulates ion transport. Proc Natl Acad Sci USA 98:11749–11754

    Article  PubMed  CAS  Google Scholar 

  24. Markwell MA, Haas SM, Bieber LL, Tolbert NE (1978) A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210

    Article  PubMed  CAS  Google Scholar 

  25. Betz WJ, Mao F, Smith CB (1996) Imaging exocytosis and endocytosis. Curr Opin Neurobiol 6:365–371

    Article  PubMed  CAS  Google Scholar 

  26. Cove J, Blinder P, Abi-Jaoude E, Lafreniere-Roula M, Devroye L, Baranes D (2006) Growth of neurites toward neurite–neurite contact sites increases synaptic clustering and secretion and is regulated by synaptic activity. Cereb Cortex 16:83–92

    Article  PubMed  Google Scholar 

  27. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16:521–555

    Article  PubMed  CAS  Google Scholar 

  28. Takagishi Y, Yasui K, Severs NJ, Murata Y (2000) Species-specific difference in distribution of voltage-gated L-type Ca(2+) channels of cardiac myocytes. Am J Physiol Cell Physiol 279:C1963–C1969

    PubMed  CAS  Google Scholar 

  29. Lipscombe D, Helton TD, Xu W (2004) L-type calcium channels: the low down. J Neurophysiol 92:2633–2641

    Article  PubMed  CAS  Google Scholar 

  30. Wu LG, Westenbroek RE, Borst JG, Catterall WA, Sakmann B (1999) Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J Neurosci 19:726–736

    PubMed  CAS  Google Scholar 

  31. Obermair GJ, Szabo Z, Bourinet E, Flucher BE (2004) Differential targeting of the L-type Ca2+ channel alpha 1C (CaV1.2) to synaptic and extrasynaptic compartments in hippocampal neurons. Eur J Neurosci 19:2109–2122

    Article  PubMed  Google Scholar 

  32. Tokunaga T, Miyazaki K, Koseki M, Mobarakeh JI, Ishizuka T, Yawo H (2004) Pharmacological dissection of calcium channel subtype-related components of strontium inflow in large mossy fiber boutons of mouse hippocampus. Hippocampus 14:570–585

    Article  PubMed  CAS  Google Scholar 

  33. Urbano FJ, Depetris RS, Uchitel OD (2001) Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals. Pflugers Arch 441:824–831

    Article  PubMed  CAS  Google Scholar 

  34. Benoff S, Goodwin LO, Millan C, Hurley IR, Pergolizzi RG, Marmar JL (2005) Deletions in L-type calcium channel alpha1 subunit testicular transcripts correlate with testicular cadmium and apoptosis in infertile men with varicoceles. Fertil Steril 83:622–634

    Article  PubMed  CAS  Google Scholar 

  35. Grunnet M, Kaufmann WA (2004) Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain. J Biol Chem 279:36445–36453

    Article  PubMed  CAS  Google Scholar 

  36. Kobayashi T, Mori Y (1998) Ca2+ channel antagonists and neuroprotection from cerebral ischemia. Eur J Pharmacol 363:1–15

    Article  PubMed  CAS  Google Scholar 

  37. Jirakulaporn T, Muslin AJ (2004) Cation diffusion facilitator proteins modulate Raf-1 activity. J Biol Chem 279:27807–27815

    Article  PubMed  CAS  Google Scholar 

  38. Illario M, Cavallo AL, Bayer KU, Di Matola T, Fenzi G, Rossi G, Vitale M (2003) Calcium/calmodulin-dependent protein kinase II binds to Raf-1 and modulates integrin-stimulated ERK activation. J Biol Chem 278:45101–45108

    Article  PubMed  CAS  Google Scholar 

  39. Dumaz N, Marais R (2003) Protein kinase A blocks Raf-1 activity by stimulating 14-3-3 binding and blocking Raf-1 interaction with Ras. J Biol Chem 278:29819–29823

    Article  PubMed  CAS  Google Scholar 

  40. Lorenz K, Lohse MJ, Quitterer U (2003) Protein kinase C switches the Raf kinase inhibitor from Raf-1 to GRK-2. Nature 426:574–579

    Article  PubMed  CAS  Google Scholar 

  41. Liuzzi JP, Cousins RJ (2004) Mammalian zinc transporters. Annu Rev Nutr 24:151–172

    Article  PubMed  CAS  Google Scholar 

  42. Palmiter RD, Cole TB, Quaife CJ, Findley SD (1996) ZnT-3, a putative transporter of zinc into synaptic vesicles. Proc Natl Acad Sci USA 93:14934–14939

    Article  PubMed  CAS  Google Scholar 

  43. MacDiarmid CW, Milanick MA, Eide DJ (2002) Biochemical properties of vacuolar zinc transport systems of Saccharomyces cerevisiae. J Biol Chem 277:39187–39194

    Article  PubMed  CAS  Google Scholar 

  44. Hell JW, Westenbroek RE, Warner C, Ahlijanian MK, Prystay W, Gilbert MM, Snutch TP, Catterall WA (1993) Identification and differential subcellular localization of the neuronal class C and class D L-type calcium channel alpha 1 subunits. J Cell Biol 123:949–962

    Article  PubMed  CAS  Google Scholar 

  45. Waters J, Smith SJ (2000) Phorbol esters potentiate evoked and spontaneous release by different presynaptic mechanisms. J Neurosci 20:7863–7870

    PubMed  CAS  Google Scholar 

  46. Petzold GC, Scheibe F, Braun JS, Freyer D, Priller J, Dirnagl U, Dreier JP (2005) Nitric oxide modulates calcium entry through P/Q-type calcium channels and N-methyl-d-aspartate receptors in rat cortical neurons. Brain Res 1063:9–14

    Article  PubMed  CAS  Google Scholar 

  47. Chen J, Daggett H, De Waard M, Heinemann SH, Hoshi T (2002) Nitric oxide augments voltage-gated P/Q-type Ca(2+) channels constituting a putative positive feedback loop. Free Radic Biol Med 32:638–649

    Article  PubMed  CAS  Google Scholar 

  48. Choi DW, Koh JY (1998) Zinc and brain injury. Annu Rev Neurosci 21:347–375

    Article  PubMed  CAS  Google Scholar 

  49. Yagami T, Ueda K, Sakaeda T, Itoh N, Sakaguchi G, Okamura N, Hori Y, Fujimoto M (2004) Protective effects of a selective L-type voltage-sensitive calcium channel blocker, S-312-d, on neuronal cell death. Biochem Pharmacol 67:1153–1165

    Article  PubMed  CAS  Google Scholar 

  50. Tsushima RG, Wickenden AD, Bouchard RA, Oudit GY, Liu PP, Backx PH (1999) Modulation of iron uptake in heart by L-type Ca2+ channel modifiers: possible implications in iron overload. Circ Res 84:1302–1309

    PubMed  CAS  Google Scholar 

  51. Oudit GY, Sun H, Trivieri MG, Koch SE, Dawood F, Ackerley C, Yazdanpanah M, Wilson GJ, Schwartz A, Liu PP, Backx PH (2003) L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med 9:1187–1194

    Article  PubMed  CAS  Google Scholar 

  52. Inoue K, Matsuda K, Itoh M, Kawaguchi H, Tomoike H, Aoyagi T, Nagai R, Hori M, Nakamura Y, Tanaka T (2002) Osteopenia and male-specific sudden cardiac death in mice lacking a zinc transporter gene, Znt5. Hum Mol Genet 11:1775–1784

    Article  PubMed  CAS  Google Scholar 

  53. Xiao GQ, Hu K, Boutjdir M (2001) Direct inhibition of expressed cardiac l- and t-type calcium channels by IgG from mothers whose children have congenital heart block. Circulation 103:1599–1604

    PubMed  CAS  Google Scholar 

  54. Dalton TP, He L, Wang B, Miller ML, Jin L, Stringer KF, Chang X, Baxter CS, Nebert DW (2005) Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis. Proc Natl Acad Sci USA 102:3401–3406

    Article  PubMed  CAS  Google Scholar 

  55. Fernandez EL, Gustafson AL, Andersson M, Hellman B, Dencker L (2003) Cadmium-induced changes in apoptotic gene expression levels and DNA damage in mouse embryos are blocked by zinc. Toxicol Sci 76:162–170

    Article  PubMed  CAS  Google Scholar 

  56. Tang W, Sadovic S, Shaikh ZA (1998) Nephrotoxicity of cadmium-metallothionein: protection by zinc and role of glutathione. Toxicol Appl Pharmacol 151:276–282

    Article  PubMed  CAS  Google Scholar 

  57. Maret W (2003) Cellular zinc and redox states converge in the metallothionein/thionein pair. J Nutr 133:1460S–1462S

    PubMed  CAS  Google Scholar 

  58. Sensi SL, Ton-That D, Sullivan PG, Jonas EA, Gee KR, Kaczmarek LK, Weiss JH (2003) Modulation of mitochondrial function by endogenous Zn2+ pools. Proc Natl Acad Sci USA 100:6157–6162

    Article  PubMed  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Michal Hershfinkel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-006-0062-4

Keywords

Navigation