Zinc is the second most abundant transition
metal in the body. Despite the fact that hundreds of biomolecules require
zinc for proper function and/or structure, the mechanism of
zinc transport into cells is not well-understood. The ZIP (Zrt- and Irt-like
proteins; SLC39A) family of
proteins acts to increase cytosolic concentrations of
zinc. Mutations in one member of the ZIP family of
proteins, the human ZIP4 (hZIP4; SLC39A4)
protein, can result in the disease
acrodermatitis enteropathica (AE). AE is characterized by growth retardation and
diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4
protein expression has been elucidated, the
cation specificity, kinetic parameters of
zinc transport, and residues involved in
cation translocation are unresolved questions. Therefore, we have established a high signal-to-noise
zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that
zinc,
copper(II), and
nickel can be transported by hZIP4 when the
cation concentration is in the micromolar range. We have also identified a nanomolar binding affinity where
copper(II) and
zinc can be transported. In contrast, under these conditions,
nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with
maleimide or
diethylpyrocarbonate indicates that extracellularly accessible
histidine, but not
cysteine, residues are required, either directly or indirectly, for
cation uptake. The results of our experiments identify at least two coordination sites for
divalent cations and provide a new framework for investigating the ZIP family of
proteins.