Abstract
The trace metal copper is an essential cofactor for a number of biological processes including mitochondrial oxidative phosphorylation, free radical detoxification, neurotransmitter synthesis and maturation, and iron metabolism. Consequently, copper transport at the cell surface and the delivery of copper to intracellular proteins are critical events in normal physiology. Little is known about the molecules and biochemical mechanisms responsible for copper uptake at the plasma membrane in mammals. Here, we demonstrate that human Ctr1 (hCtr1) is a component of the copper transport machinery at the plasma membrane. hCtr1 transports copper with high affinity in a time-dependent and saturable manner and is metal-specific. hCtr1-mediated (64)Cu transport is an energy-independent process and is stimulated by extracellular acidic pH and high K(+) concentrations. hCtr1 exists as a homomultimer at the plasma membrane in mammalian cells. This is the first report on the biochemical characterization of the human copper transporter hCtr1, which is important for understanding mechanisms for mammalian copper transport at the plasma membrane.
Highlights
Copper is a micronutrient that plays an essential role in biology, serving as a co-factor for several enzymes that include Cu,Zn-superoxide dismutase, cytochrome oxidase, lysyl oxidase, and ceruloplasmin [1, 2]
To ascertain whether human Ctr1 (hCtr1) plays a role in copper transport in human cells, hCtr1 protein was expressed in Hek293 cells by transient transfection of an expression plasmid carrying the hCtr1 open reading frame under the control of the cytomegalovirus promoter
The initial rate of copper uptake was measured in Hek293 cells expressing hCtr1 by incubation of the cells with 2 M 64Cu for 5 min, and the effects of potential copper ligands in serum, such as albumin and histidine, on hCtr1-stimulated 64Cu uptake were examined. 64Cu accumulation in the vector control cells indicates the presence of a low level of copper uptake in Hek293 cells (Fig. 1), consistent with low but detectable levels of hCtr1 mRNA detected in these cells by RNA blotting analysis. hCtr1 expression stimulates the initial rate of 64Cu uptake 3–7-fold over vectortransfected control cells, depending on the transfection efficiency in each experiment (Fig. 1)
Summary
Copper is a micronutrient that plays an essential role in biology, serving as a co-factor for several enzymes that include Cu,Zn-superoxide dismutase, cytochrome oxidase, lysyl oxidase, and ceruloplasmin [1, 2]. Studies in yeast cells first identified genes encoding high affinity copper ion transport proteins in the plasma membrane. We have examined the kinetics of copper transport, metal specificity, localization, oligomerization, and roles for energy and an electrochemical gradient at the plasma membrane in copper uptake by the functional expression of hCtr11 in a human embryonic kidney (Hek293) cell line.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
