Functional Transport Properties of Human Zinc Transporter 1: Kinetics and pH-Dependency

Intracellular zinc (Zn²⁺) homeostasis is essential for physiological and pathological processes and is strictly regulated by Zn²⁺ transporters. Zinc transporter 1 (ZnT1) is a ubiquitously expressed plasma membrane-localized Zn transporter that exports Zn²⁺ from the cytoplasm to the extracellular space. However, the functional transport properties regarding kinetics and driving forces of ZnT1 remain debatable. In this study, we established a cell-free proteoliposome assay system and demonstrated that ZnT1 transports Zn²⁺ with high affinity in pH-dependent and pH-independent manners. The K_m and V_max of pH-dependent Zn²⁺ transport were 0.40 μM and 15.13 nmol/min/mg protein, and those of pH-independent Zn²⁺ transport were 0.52 μM and 8.88 nmol/min/mg protein (low concentrations of Zn²⁺), 3.02 μM and 17.59 nmol/min/mg protein (high concentrations of Zn²⁺), respectively, suggesting biphasic kinetic components of Zn²⁺ transport. Even without pH gradient formation, ZnT1 exhibits potent Zn²⁺ transport activity. In pH dependency, Zn²⁺ transport activity was higher at an inside pH of 6.0 than at 6.5-7.5 for proteoliposomes, despite the same ΔpH of 0.5-1.5. The Zn²⁺ transport activity decreased at an outside pH of 8.0, despite an increase in ΔpH. Although previous studies have proposed that ZnT1-mediated Zn²⁺ transport activity is driven by a calcium (Ca²⁺) gradient and not by a pH gradient, Ca²⁺ does not enhance Zn²⁺ transport activity in the presence or absence of a pH gradient. These results strongly suggest that ZnT1 protein transports Zn²⁺ optimally at a specific pH and exports excess intracellular Zn²⁺ even without ΔpH.