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Intracellular zinc levels are strictly regulated by zinc channels and zinc-binding proteins to maintain cellular zinc-dependent functions. We demonstrated a correlation between extracellular zinc concentration and intracellular exchangeable zinc levels using the fluorescent zinc-specific probes zinquin and zinpyr-1. The effect of extracellular zinc status on the regulation of the two trans-Golgi network directed zinc transporters ZnT-5 and ZnT-7 was next studied by real-time RT-PCR in zinc supplemented or depleted HeLa cells. While sub-toxic extracellular zinc addition strongly induced the efflux transporter ZnT-1 gene expression, consistent with its activation by the transcription factor MTF-1, treated HeLa cells did not display any change in ZnT-5 and ZnT-7 mRNA levels compared to control cells. In contrast, zinc depletion induced by non-toxic doses of the zinc chelator TPEN (N,N,N',N' tetrakis-(2 pyridylmethyl) ethylene diamine) resulted in a up to eight-fold induction of transporters ZnT-5 and ZnT-7 mRNA levels, providing the first evidence of a transcriptional control of these two zinc efflux transporters by zinc deficiency in cultured cells.