Cell-based transport assay to study kinetics and substrate specificity of human ZIPs

Kinetic study of human ZIPs is crucial for understanding the transport mechanism and the molecular basis of substrate specificity. In this chapter, we describe the detailed experimental procedures for functional studies of two human ZIPs, including the zinc-preferring ZIP4 and the multi-metal transporter ZIP8, by using the cell-based transport assays. Kinetic study of ZIP4 is elaborated in the first section; in the second section, comparison of ZIP4 and ZIP8 in terms of the zinc/cadmium selectivity is performed by using an internal competition assay adapted from the established cell-based approach. The protocols provided in this chapter will facilitate mechanistic and engineering studies of the ZIPs.