The effect of divalent metal cations on zinc uptake by mouse Zrt/Irt-like protein 1 (ZIP1)

Aim: In this study, we evaluated the effect of divalent metal cations on zinc uptake via mouse Zrt/Irt-like protein 1 (mZIP1), a ubiquitously expressed zinc transporter, which plays a role in maintenance of cellular zinc homeostasis, using HEK293T cells overexpressing it. Main methods: mZIP1 cDNA, which was cloned from mouse microglia, was transfected into HEK293T cells by a lipofection method, and its functional expression was confirmed by Western blotting and immunocytochemical analyses, and Zn-65 ((ZnCl2)-Zn-65) uptake. Key findings: Zn-65 uptake by mZIP1 cDNA-transfected cells time-dependently increased compared with that by mock cells, indicating functional expression of mZIP1. mZIP1-mediated Zn-65 uptake showed clear saturable kinetics consisting of a single component with a Michaelis constant of 5.88 mu M. FeCl2 and NiCl2 competitively inhibited the Zn-65 uptake, the inhibition constants (K-i) being estimated to be 0.92 and 28.6 mu M, respectively. In addition, CoCl2 and CdCl2 showed non-competitive inhibition of mZIP1-mediated Zn-65 uptake, the K-i values being 219 and 32.5 mu M, respectively. On the other hand, CuCl2 also significantly decreased the uptake, but the inhibition mode could not evaluate because of its low solubility, while MnCl2 and MgCl2 had no effect on Zn-65 uptake via mZIP1. Significance: Iron, nickel, cobalt and cadmium act as inhibitors of mZIP1, the affinity order being iron > zinc > nickel = cadmium > cobalt, and copper might also act as an inhibitor, while manganese and magnesium are not recognized by mZIP1. These findings provide valuable information as to the contribution of mZIP1 to total cellular zinc transport. (C) 2014 Elsevier Inc. All rights reserved.