Inhibitory effect of divalent metal cations on zinc uptake via mouse Zrt-/Irt-like protein 8 (ZIP8)

Aims: There is controversy regarding the substrate specificity of ZIP8, a ZIP isoform, involved in regulation of extra- and intracellular zinc levels. Here, we investigated the inhibitory effects of divalent metal cations on zinc uptake via mouse ZIP8 (mZIP8). Main methods: mZIP8 cDNA was transfected into HEK293T cells by a lipofection method, and its functional expression was evaluated by immunocytochemistry, Western blotting and Zn-65 ((ZnCl2)-Zn-65) uptake measurement. Key findings: Transfection of mZIP8 cDNA into HEK293T cells induced expression of mZIP8 in the cells, and increased zinc uptake. mZIP8-mediated zinc uptake depended on extracellular bicarbonate, and the Michaelis constant for the uptake was estimated to be 8.48 +/- 2.46 mu M. In the inhibition study, iron and cadmium competitively, and cobalt, nickel and copper non-competitively inhibited the mZIP8-mediated zinc uptake, the inhibition constants being calculated to be 3.37, 55.5, 80.6, 198 and 48.3 mu M, respectively. In contrast, magnesium and manganese at concentrations of up to 1500 and 200 mu M, respectively, had no inhibitory effect on the zinc uptake via mZIP8. Significance: In this study, we reveal that the inhibition profiles of divalent metal cations as to zinc uptake via mZIP8 apparently differ from those for mZIP1, especially in the affinity and inhibition manner of nickel. These findings should contribute to identification of ZIP isoforms involved in total cellular zinc transport (C) 2016 Elsevier Inc. All rights reserved.