Rate and regulation of copper transport by human copper transporter 1 (hCTR1).

Human copper transporter 1 (hCTR1) is a homotrimer of a 190-amino acid monomer having three transmembrane domains believed to form a pore for copper permeation through the plasma membrane. The hCTR1-mediated copper transport mechanism is not well understood, nor has any measurement been made of the rate at which copper ions are transported by hCTR1. In this study, we estimated the rate of copper transport by the hCTR1 trimer in cultured cells using (64)Cu uptake assays and quantification of plasma membrane hCTR1. For endogenous hCTR1, we estimated a turnover number of about 10 ions/trimer/s. When overexpressed in HEK293 cells, a second transmembrane domain mutant of hCTR1 (H139R) had a 3-fold higher Km value and a 4-fold higher turnover number than WT. Truncations of the intracellular C-terminal tail and an AAA substitution of the putative metal-binding HCH C-terminal tripeptide (thought to be required for transport) also exhibited elevated transport rates and Km values when compared with WT hCTR1. Unlike WT hCTR1, H139R and the C-terminal mutants did not undergo regulatory endocytosis in elevated copper. hCTR1 mutants combining methionine substitutions that block transport (M150L,M154L) on the extracellular side of the pore and the high transport H139R or AAA intracellular side mutations exhibited the blocked transport of M150L,M154L, confirming that Cu(+) first interacts with the methionines during permeation. Our results show that hCTR1 elements on the intracellular side of the hCTR1 pore, including the carboxyl tail, are not essential for permeation, but serve to regulate the rate of copper entry.

Cellular glutathione plays a key role in copper uptake mediated by human copper transporter 1.

Copper is an essential micronutrient. Following entry via the human copper transporter 1 (hCTR1), copper is delivered to several copper chaperones, which subsequently transfer the metal to specific targets via protein:protein interactions. It is has been assumed, but not demonstrated, that chaperones acquire copper directly from hCTR1. However, some reports have pointed to an intermediary role for glutathione (GSH), an abundant copper-binding tri-peptide. To address the issue of how transported copper is acquired by the copper chaperones in vivo, we measured the initial rate of (64)Cu uptake in cells in which the cellular levels of copper chaperones or GSH were substantially depleted or elevated. Knockdown or overexpression of copper chaperones ATOX1, CCS, or both had no effect on the initial rate of (64)Cu entry into HEK293 cells having endogenous or overexpressed hCTR1. In contrast, depleting cellular GSH using L-buthionine-sulfoximine (BSO) caused a 50% decrease in the initial rate of (64)Cu entry in HEK293 cells and other cell types. This decrease was reversed by washout of BSO or GSH replenishment with a permeable ester. BSO treatment under our experimental conditions had no significant effects on the viability, ATP levels, or metal content of the cells. Attenuated (64)Cu uptake in BSO was not due to oxidation of the cysteine in the putative metal-binding motif (HCH) at the intracellular hCTR1 COOH terminus, because a mutant lacking this motif was fully active, and (64)Cu uptake was still reduced by BSO treatment. Our data suggest that GSH plays an important role in copper handling at the entry step.

Copper-dependent recycling of hCTR1, the human high affinity copper transporter.

Copper is an essential co-factor in many important physiological processes, but at elevated levels it is toxic to cells. Thus at both the organism and cellular level mechanisms have evolved to finely tune copper homeostasis. The protein responsible for copper entry from the circulation in most human cells is hCTR1, a small protein (190 amino acid residues) that functions as a trimer in the plasma membrane. In the present work we employ cell surface biotinylation and isotopic copper uptake studies of overexpressed hCTR1 in HEK293 cells to examine the acute (minutes) response of hCTR1 to changes in extracellular copper. We show that within 10 min of exposure to copper at 2.5 microM or higher, plasma membrane hCTR1 levels are reduced (by approximately 40%), with a concomitant reduction in copper uptake rates. We are unable to detect any degradation of internalized hCTR1 in the presence of cycloheximide after up to 2 h of exposure to 0-100 microM copper. Using a reversible biotinylation assay, we quantified internalized hCTR1, which increased upon the addition of copper and corresponded to the hCTR1 lost from the surface. In addition, when extracellular copper is then removed, internalized hCTR1 is promptly (within 30 min) recycled to the plasma membrane. We have shown that in the absence of added extracellular copper, there is a small but detectable amount of internalized hCTR1 that is increased in the presence of copper. Similar studies on endogenous hCTR1 show a cell-specific response to elevated extracellular copper. Copper-dependent internalization and recycling of hCTR1 provides an acute and reversible mechanism for the regulation of cellular copper entry.

Urinary copper elevation in a mouse model of Wilson's disease is a regulated process to specifically decrease the hepatic copper load.

Body copper homeostasis is regulated by the liver, which removes excess copper via bile. In Wilson's disease (WD), this function is disrupted due to inactivation of the copper transporter ATP7B resulting in hepatic copper overload. High urinary copper is a diagnostic feature of WD linked to liver malfunction; the mechanism behind urinary copper elevation is not fully understood. Using Positron Emission Tomography-Computed Tomography (PET-CT) imaging of live Atp7b(-/-) mice at different stages of disease, a longitudinal metal analysis, and characterization of copper-binding molecules, we show that urinary copper elevation is a specific regulatory process mediated by distinct molecules. PET-CT and atomic absorption spectroscopy directly demonstrate an age-dependent decrease in the capacity of Atp7b(-/-) livers to accumulate copper, concomitant with an increase in urinary copper. This reciprocal relationship is specific for copper, indicating that cell necrosis is not the primary cause for the initial phase of metal elevation in the urine. Instead, the urinary copper increase is associated with the down-regulation of the copper-transporter Ctr1 in the liver and appearance of a 2 kDa Small Copper Carrier, SCC, in the urine. SCC is also elevated in the urine of the liver-specific Ctr1(-/-) knockouts, which have normal ATP7B function, suggesting that SCC is a normal metabolite carrying copper in the serum. In agreement with this hypothesis, partially purified SCC-Cu competes with free copper for uptake by Ctr1. Thus, hepatic down-regulation of Ctr1 allows switching to an SCC-mediated removal of copper via kidney when liver function is impaired. These results demonstrate that the body regulates copper export through more than one mechanism; better understanding of urinary copper excretion may contribute to an improved diagnosis and monitoring of WD.

O-linked glycosylation at threonine 27 protects the copper transporter hCTR1 from proteolytic cleavage in mammalian cells.

The major human copper uptake protein, hCTR1, has 190 amino acids and a predicted mass of 21 kDa. hCTR1 antibodies recognize multiple bands in SDS-PAGE centered at 35 kDa. Part of this increased mass is due to N-linked glycosylation at Asn-15. We show that in mammalian cells the N15Q mutant protein trafficked to the plasma membrane and mediated copper uptake at 75% of the rate of wild-type hCTR1. We demonstrate that the extracellular amino terminus of hCTR1 also contains O-linked polysaccharides. Glycosidase treatment that removed O-linked sugars reduced the apparent mass of hCTR1 or N15Q mutant protein by 1-2 kDa. Expression of amino-terminal truncations and alanine substitution mutants of hCTR1 in HEK293 and MDCK cells localized the site of O-linked glycosylation to Thr-27. Expression of alanine substitutions at Thr-27 resulted in proteolytic cleavage of hCTR1 on the carboxyl side of the T27A mutations. This cleavage produced a 17-kDa polypeptide missing approximately the first 30 amino acids of hCTR1. Expression of wild-type hCTR1 in mutant Chinese hamster ovary cells that were unable to initiate O-glycosylation also resulted in hCTR1 cleavage to produce the 17-kDa polypeptide. The 17-kDa hCTR1 polypeptide was located in the plasma membrane and mediated copper uptake at about 50% that of the rate of wild-type hCTR1. Thus, O-linked glycosylation at Thr-27 is necessary to prevent proteolytic cleavage that removes half of the extracellular amino terminus of hCTR1 and significantly impairs transport activity of the remaining polypeptide.

Copper entry into human cells: progress and unanswered questions.

In this brief review we summarize what is known about the role of hCTR1 in mediating the entry of copper into human cells. There is a body of information that clearly identifies this protein as being a major source (though not the only source) of copper entry into human cells, and thus a crucial element of copper homeostasis. However, much remains that is poorly understood and key aspects of the physiological roles of hCTR1 and its regulation are only superficially appreciated. The particular characteristics of a transport process that in vivo involves the binding, transmembrane transport and release of a substrate that is not present in a free form in the intracellular or extracellular compartments poses particular challenges that are not encountered in the transport of more familiar physiologically important metal cations. Thus much of what we have learned about the more commonly encountered transported ions provides an inadequate model for studies of copper homeostasis. In this article we review progress made and identify the major questions that need to be resolved before an adequate description is attained of how copper entry into human cells is mediated and regulated by hCTR1.