Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn²⁺ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn²⁺ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn²⁺. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn²⁺ levels. With the development of intracellular Zn²⁺ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn²⁺, detected using fluorescent probes, with the expression of the Zn²⁺ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn²⁺ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn²⁺ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn²⁺ by utilizing the Zn²⁺ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn²⁺. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.