The Role of the Methionines and Histidines in the Transmembrane Domain of Mammalian Copper Transporter 1 in the Cellular Accumulation of Cisplatin

Abstract

Mammalian copper transporter 1 (CTR1) is a high-affinity copper influx transporter that also mediates the uptake of platinum-containing chemotherapeutic agents including cisplatin (cDDP). Methionines 150, 154, and histidine 139 have been proposed to form a series of stacked rings in the pore formed by the CTR1 homotrimer, each of which is required for maximal copper transport. To examine the mechanism by which hCTR1 also transports cDDP, variant forms of hCTR1 in which methionines 150 and 154 were converted to isoleucines or in which histidine 139 was converted to alanine were re-expressed in cells in which both alleles of CTR1 had been knocked out. Each of these conversions disabled copper transport and increased cellular resistance to the cytotoxic effect of copper. In contrast, conversion of the methionines increased the uptake and cytotoxicity of cDDP well above that attained with wild-type hCTR1. Conversion of His139 to alanine did not impair cDDP uptake and actually enhanced cytotoxicity.

Ctr2 regulates biogenesis of a cleaved form of mammalian Ctr1 metal transporter lacking the copper- and cisplatin-binding ecto-domain

“…Moreover, Ctr1 plays an important role in cellular uptake of cisplatin, and the levels of Ctr1 influence cisplatin accumulation in cellular and cancer models (25,29,35,51). Here we present data that support a role for Ctr2 in regulating copper and cisplatin acquisition via Ctr1.…”

“…In contrast to Cu + uptake, the Met-rich ectodomain of yeast Ctr1 is required for cisplatin import (27). Moreover, as Ctr1 M-X 3 -M mutants are competent for cisplatin uptake, but not Cu + (35), studies suggest that Ctr1-mediated cisplatin uptake may occur via an ecto-domain-dependent receptor-mediated endocytosis mechanism, rather than as an ion channel as for Cu + (27). Ctr1 has been observed in both cell lines and mouse tissues as a full-length glycosylated form and a lowermolecular-weight form, which has been reported to lack a portion of the Cu + and cisplatin-binding ecto-domain (17,36).…”

A Re-Evaluation of the Role of hCTR1, the Human High-Affinity Copper Transporter, in Platinum-Drug Entry into Human Cells

“…In previous studies using mefs, in contrast to our results, higher uptake of Pt was seen in cells expressing hCTR1 than in 2/2 cells (Larson et al, , 2010a. These studies make several interesting points: most of the overexpressed hCTR1 protein was not delivered to the plasma membrane, there is a lack of correlation between cDDP uptake and cytotoxicity, truncation of the first 45 amino acids of hCTR1 does not inhibit cDDP uptake (Larson et al, 2010b), and replacement of Met residues 150 and 154 (Larson et al, 2010a) increases cDDP uptake and decreases Cu uptake. The authors conclude Cisplatin Entry into Human Cells that if hCTR1 is responsible for cDDP uptake, its interactions with cDDP are different from those with Cu.…”

Ionization Properties of Histidine Residues in the Lipid Bilayer Membrane Environment

“…It is therefore vitally important to also know the ionization properties of the His side chain at various locations within lipid bilayer membranes. Indeed, as with soluble proteins, many membrane proteins contain functionally important His residues within their transmembrane domains: for example, those of the photosynthetic reaction center (4), cytochrome c oxidase (5,6), influenza A M2 channel (7)(8)(9)(10), and other transporters and channels (11)(12)(13)(14). Given that proton conduction may require clusters of multiple residues (5,6), it is important to know the limits for the titration properties of candidate residues such as histidines and carboxyl groups, among others, in bilayer membranes.…”