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Comparative Study
. 2008 Jan 25;365(4):814-20.
doi: 10.1016/j.bbrc.2007.11.067. Epub 2007 Nov 26.

Cd2+ Versus Zn2+ Uptake by the ZIP8 HCO3--dependent Symporter: Kinetics, Electrogenicity and Trafficking

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Free PMC article
Comparative Study

Cd2+ Versus Zn2+ Uptake by the ZIP8 HCO3--dependent Symporter: Kinetics, Electrogenicity and Trafficking

Zhiwei Liu et al. Biochem Biophys Res Commun. .
Free PMC article

Abstract

The mouse Slc39a8 gene encodes the ZIP8 transporter, which has been shown to be a divalent cation/HCO3- symporter. Using ZIP8 cRNA-injected Xenopus oocyte cultures, we show herein that: [a] ZIP8-mediated cadmium (Cd(2+)) and zinc (Zn(2+)) uptake have V(max) values of 1.8+/-0.08 and 1.0+/-0.08 pmol/oocyte/h, and K(m) values of 0.48+/-0.08 and 0.26+/-0.09 microM, respectively; [b] ZIP8-mediated Cd(2+) uptake is most inhibited by Zn(2+), second-best inhibited by Cu(2+), Pb(2+) and Hg(2+), and not inhibited by Mn(2+) or Fe(2+); and [c] electrogenicity studies demonstrate an influx of two HCO3- anions per one Cd(2+) (or one Zn(2+)) cation, i.e. electroneutral complexes. Using Madin-Darby canine kidney (MDCK) polarized epithelial cells retrovirally infected with ZIP8 cDNA and tagged with hemagglutinin at the C-terminus, we show that-similar to ZIP4-the ZIP8 eight-transmembrane protein is largely internalized during Zn(2+) homeostasis, but moves predominantly to the cell surface membrane (trafficking) under conditions of Zn(2+) depletion.

Figures

FIG. 1
FIG. 1
Kinetics of cation uptake in Xenopus laevis oocytes. A, top, Cd uptake as a function of time in ZIP8 cRNA-injected versus water-injected oocytes. *P <0.01. **P <0.001. At bottom, Cd uptake as a function of its concentration in ZIP8 cRNA-injected minus water-injected oocytes; incubation time was 60 min. B, top, Zn uptake in ZIP cRNA-injected versus water-injected oocytes. *P <0.05. **P <0.01. At bottom, Zn uptake, determined as described in part A. Circles and brackets at top denote means ± S.E. Vmax and Km values are expressed as means ± S.E. Circles and brackets at bottom denote means ± S.D.
FIG. 2
FIG. 2
Metal cation competition for Cd uptake in Xenopus oocytes. Non-labeled Cd was spiked with 109CdCl2 to make a final Cd concentration of 0.25 μM; the competing metal cations at concentrations of 0, 0.75, 2.5 or 7.5 μM were added at the same time as the Cd, and the oocytes were incubated at 20°C for 60 min, following which Cd accumulation was determined. *P <0.001. **P < 0.01).
FIG. 3
FIG. 3
Electrogenicity studies. A, top, Cd (0.5 μM) uptake by water-injected oocytes. At bottom, Cd uptake by ZIP8 cRNA-injected, minus water-injected oocytes. B, Zn (1.0 μM) uptake by water-injected oocytes (top) and by ZIP8 cRNA-injected, minus water-injected oocytes (bottom). Note the ordinates for each of the four panels are different. Incubation time 30 min.
FIG. 4
FIG. 4
A, Western immunoblot to detect the relative amounts of ZIP4ha versus ZIP8ha protein on the surface of MDCK cells, carried out identically to that previously described [24]. Equal loading was confirmed by Coomassie staining (not shown). B, Schematic diagram showing location of the membrane-bound ZIP8 transporter under normal Zn homeostasis (left), compared with that under low zinc conditions (right).

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