Simple in vitro method to evaluate ZIP zinc transport ability through zinc transporter 1 and metallothionein expression measurements.

Abstract

Measuring the cellular zinc content and examining the alteration of zinc status are critical for investigating the cellular homeostasis and dynamics of zinc and its involvement in patho-physiological functions. Many Zrt- and Irt-related protein (ZIP) transporters uptake zinc from the extracellular space. Among Zn transporters (ZNTs), ZNT1 effluxes cytosolic zinc. As cytosolic zinc-binding proteins, metallothioneins (MTs) also contribute to the control of cellular zinc homeostasis. Systemic and cellular zinc homeostasis is considered to be maintained by balancing expression and functional activities of these proteins. The zinc transport ability of ZIPs is typically measured by evaluating cellular zinc content with various zinc-detection methods and systems. Many small-molecule fluorescent probes and fluorescence resonance energy transfer-based protein sensors have been exploited for this purpose. Although powerful analytical methods using special instruments have been developed to quantify zinc, they are often not easily accessible. Here, we present a simplified and inexpensive method to estimate the zinc transport ability of ZIP transporters using the expression responses of ZNT1 and MT. This protocol should be effective in several applications because ZNT1 and MT expression are easily evaluated by immunoblotting and immunofluorescence staining as basic biochemical techniques available in most laboratories. This method is advantageous for examining the relative zinc status or alterations mediated by expression changes of ZIPs in cells cultured in normal medium without zinc supplementation. As zinc is an essential micronutrient, extensive research is necessary to improve dietary zinc absorption to promote health. Therefore, we also propose a simple screening method of foods to improve zinc absorption as an application of measuring ZIP-mediated MT expression.