Copper uptake mechanisms in epithelial cells

The goal of this research was to characterize copper transport and the localization of endogenous copper transporter CTR1 in polarized intestinal Caco2 and T84 cells, and renal epithelial MDCK and OK cells.;Results obtained from these investigations clearly support the presence of functional CTR1 at the basolateral membrane in all the epithelial cell lines studied, as well as in intestinal tissue. This finding was unexpected as CTR1, the only identified high affinity copper uptake system in mammals, was postulated to mediate absorption of dietary copper from the gut, implying localization at the apical surface of intestinal cells. However, the evidence presented in this thesis supports localization of hCTR1 at the basolateral surface, where it mediates copper uptake from the blood. This is in agreement with the recently published study of transgenic mice with a conditional knockout of ctr1 gene in intestine. The ctr1-/- phenotype was characterized by an accumulation of copper in enterocytes, rather than copper deficiency, clearly showing that CTR1 is not directly involved in the process of apical Cu uptake. Other observations also suggested that alternative mechanisms of copper uptake must exist.;The goal of the second project was to explore such alternative mechanisms of copper transport which might be responsible for the acquisition of copper from diet. More specifically, the possibility of sodium-coupled amino acid transporter taking part in uptake of copper as copper-bis-histidine complexes was excluded, and the mechanism for the high copper fluxes that occur when histidine, albumin, or serum are absent from the transport medium, was characterized. The basis for the latter mechanism is formation of copper-anion complexes, and it may be an important contributor to the apical acquisition of dietary copper from the intestinal lumen, suggesting a new role for the SLC26A family of anion exchangers in copper transport.;Finally, in preliminary studies utilizing an intracellular fluorescent indicator, calcein, evidence is presented that copper entry and subsequent handling within the cell depends on the oxidation state of the transported copper.