ZnT‐1 protein expression or localization does not explain effects of reduced availability of zinc on cellular zinc efflux

Abstract

Reducing zinc availability to H4IIE rat hepatoma cells with the extracellular chelator DTPA (diethylenetriaminepentaacetic acid) results in the rapid inhibition of cellular zinc efflux. In contrast, DTPA increases zinc efflux in primary hepatocytes. The SLC30 or ZnT group of membrane transporters is responsible for reducing cytoplasmic zinc, both via cellular efflux and organelle uptake. ZnT‐1 is believed to be key for zinc efflux, due to its reported plasma membrane localization. However, we found that DTPA does not change the concentration of ZnT‐1 mRNA or protein in H4IIE cells. Since cells could also control efflux by shifting the cellular location of ZnT‐1, we incubated H4IIE cells and primary hepatocytes with DTPA (50μM) or zinc sulfate (100 μM) for 2 hours and examined ZnT‐1 localization using immunocytochemistry. The distribution of ZnT‐1 immunofluorescence, which was observed throughout the cytoplasm, did not change with treatment in either cell type. We also treated H4IIE cells with DTPA or zinc and isolated plasma membrane proteins, following biotinylation and avidin purification. Neither DTPA nor zinc affected the amount of ZnT‐1 recovered in the biotinylated fraction as assessed by Western analysis. Thus we conclude that the rapid homeostatic response of cells to altered zinc availability must be attributed to a transporter other than ZnT‐1 or to changes in the activity of ZnT‐1 by a novel mechanism.