Effects of copper availability on Ctr1 expression in B104 rat neuroblastoma cell line

Aim: Copper is an essential trace metal required as a cofactor in a broad range of enzymatic functions. Copper transporter-1 (Ctr1), ubiquitously expressed, is believed to play a major role in Cu uptake into the cell. In the present study we used a rat neuroblastoma cell line (B104) as a cell model aiming to examine how extracellular copper availability can modulate the expression of Ctr1 and Cu fluxes. Methods: B104 cells were cultured under Cu depletion by exposure to cuprizone, a specific Cu chelating agent, and Cu supplementation for rising time periods (48, 96 hours). Ctr1 protein levels were assessed by Western Blotting analysis. Changes in transmembrane copper fluxes were measured by a fluorimetric method employing the Cu+-sensitive fluorophore Phen Green SK. Results: Treatment of cells with 50 µM CuCl2 for 48 h determined a significant increase in Ctr1 expression, while Cu supplementation prolonged for 96 h was found to restore basal protein levels. Exposure to over-physiological Cu level didn’t alter ion transmembrane fluxes as measured by the fluorescence method, indicating that excess protein was unfunctional. Analogously to Cu supplementation, exposure of cells to ion depletion for 48 h induced an increase in protein synthesis, probably as an immediate adaptive response to inadequate copper levels in basal culture medium. No change in protein expression was detected after 96 h treatment. Conclusion: In summary, our results suggest that short-term adapting strategies of B104 cells to varying Cu availability in culture medium involve directly Ctr1, even if we are not able to exclude other possibilities. For a longer time period treatment, presumably other mechanisms can be involved, such as Ctr1 co-regulation with other ion transporters (i.e. DMT1, Divalent Metal Transporter 1).