THE PRODUCTION OF THE SMALL COPPER CARRIER AND INVESTIGATION INTO ITS STRUCTURE, AND UPTAKE BY CULTURED CELLS

Wilson disease results in excess copper (Cu) build up in the liver stopping secretion into the bile for excretion. Instead, some of the excess Cu is removed in the urine where a small Cu carrier (SCC) was identified. The goals of our research were to produce and purify SCC in cultured cells, to determine its structure, and to investigate if SCC-Cu was taken up by cells and if so how. SCC was produced by hepatic and intestinal cells, which (in the case of rat hepatoma cells) was enhanced by preloading them with Cu. It was partially purified by 3 kDa ultrafiltration, dialysis, and small pore size exclusion chromatography (SEC). NMR data of the purified sample showed that there was at least one Cu containing component among contaminants. SCC-Cu was found to be taken up by all cells tested, as shown by a linear rate of uptake from SCC-⁶⁷Cu over 5 h. Since it was readily taken up by cells we investigated methods potentially involved in the uptake. There was reduced uptake of ⁶⁷Cu-labeled SCC when the high affinity Cu uptake protein 1 (CTR1) was inhibited by excess silver (Ag) ions, which competes with Cu(I) for uptake by CTR1. Excess Cu(I) competed with uptake of SCC-⁶⁷Cu and when CTR1 was knocked out there was a markedly reduced rate of uptake. However, there was still uptake of SCC-⁶⁷Cu by cells where CTR1 had been knocked out implying CTR1 is not the only transporter involved. High concentrations of Cu(I) completely inhibited uptake of Cu from SCC-⁶⁷Cu in CTR1 knockout cells suggesting another form of uptake also involves Cu(I). Cu(II) almost completely abolished uptake and was toxic to the cells, suggesting there is an alternate Cu(II) uptake transporter. Inhibition of divalent metal transporter-1 (DMT1) did not reduce uptake of SCC-⁶⁷Cu indicating it is not involved. Inhibition of endocytosis also had no effect. Based on this evidence SCC’s function is the transport of Cu for the purpose of Cu homeostasis.