Silencing of ZnT-1 expression enhances heavy metal influx and toxicity.
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 Ca(2+) 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 Ca(2+) influx via LTCC by approximately fourfold. Moreover, ZnT-1 siRNA transfected neurons showed approximately 30% increase in synaptic release, monitored using the FM1-43 dye. An increased cation influx rate, through the LTCC, was also recorded for Zn(2+) and Cd(2+) in cells treated with the ZnT-1 siRNA. Furthermore, Cd(2+)-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 Cd(2+) influx rate via the LTCC. Finally, a robust nimodipine-sensitive Cd(2+) influx was observed using a low extracellular Cd(2+) concentration (5 muM) 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 Cd(2+) and Zn(2+) permeation and toxicity in neurons and other cell types.
Ohana
Ehud
E
Department of Physiology, Faculty of Health Sciences, Ben Gurion University of the Negev, Beer-Sheva, 84105, Israel.
Sekler
Israel
I
Kaisman
Tehila
T
Kahn
Nicol
N
Cove
Joshua
J
Silverman
William F
WF
Amsterdam
Abraham
A
Hershfinkel
Michal
M
D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects; D002104:Cadmium / Q000378:metabolism / Q000633:toxicity; D002118:Calcium / Q000378:metabolism / Q000633:toxicity; D020746:Calcium Channels, L-Type / Q000378:metabolism; D027682:Cation Transport Proteins; D005260:Female; D020868:Gene Silencing; D006107:Granulosa Cells / Q000166:cytology / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D019216:Metals, Heavy / Q000378:metabolism* / Q000633:toxicity*; D051379:Mice; D009206:Myocardium / Q000166:cytology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000473:pathology; D018377:Neurotransmitter Agents / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D014162:Transfection; D015032:Zinc / Q000378:metabolism / Q000633:toxicity
D020746:Calcium Channels, L-Type; D027682:Cation Transport Proteins; D008565:Membrane Proteins; D019216:Metals, Heavy; D018377:Neurotransmitter Agents; D034741:RNA, Small Interfering; D002104:Cadmium; C092392:Slc30a1 protein, rat; D015032:Zinc; D002118:Calcium