Mechanistic Comparison of Human High-Affinity Copper Transporter 1-Mediated Transport between Copper Ion and Cisplatin

Abstract

The human high-affinity copper transporter (hCtr1) plays an important role in the regulation of intracellular copper homeostasis. hCtr1 is involved in the transport of platinum-based antitumor agents such as cisplatin (CDDP); however, the mechanisms that regulate hCtr1-mediated transport of these agents have not been well elucidated. We compared the mechanisms of hCtr1-mediated transport of copper and CDDP. We found that replacements of several methionine residues that are essential for hCtr1-mediated copper transport conferred a dominant-negative effect on the endogenous hCtr1’s function, resulting in reduced rates of Cu(I) and CDDP transport and increased resistance to the toxicities of copper and CDDP treatments. Kinetic constant analyses revealed that although these mutations reduced maximal transport rates (V_max) for Cu(I) and CDDP, reduction of K_m only for Cu(I) but not for CDDP was observed. Mutation in Gly167, which is located in the third transmembrane domain and is involved in helix packing of hCtr1, also conferred dominant-negative property of Cu(I) transport but not of CDDP transport. Deleting the N-terminal 45 amino acids that contain two methionine-rich motifs resulted in cytoplasmic localization of the hCtr1 and abolished the dominant-negative function of these mutants. Nonetheless, these mutations did not affect the capacities of hCtr1 oligomerization induced by copper or CDDP, suggesting a distinct structural requirement between metal transport and oligomerization. Finally, we also observed that expressing the dominant-negative hCtr1 mutants up-regulates endogenous hCtr1 mRNA expression, consistent with our previous report that intracellular copper homeostasis and homeostatic levels of hCtr1 mRNA are mutually regulated.