Involvement of chloride channels in hepatic copper metabolism: ClC-4 promotes copper incorporation into ceruloplasmin

Abstract

Background & Aims: Copper transport in hepatocytes is regulated by the interaction of multiple pumps, chaperones, and accessory proteins. Intracellular chloride channels are essential for copper metabolism in yeast but their role in Cu transport in hepatocytes is unknown. The aim of this study was to determine whether chloride channels are modulators of copper incorporation into ceruloplasmin (CP). Methods: The effects of chloride concentration and chloride channel expression on secretion of holoCp and apoCp was measured by gel electrophoresis and immunoblotting. ClC family chloride channel expression in hepatocytes was determined by Western blotting. The association of ClC-4 and the Wilson’s disease protein (ATP7B) was determined by co-immunoprecipitation. Results: Chloride substitution reduced total Cp secretion and the ratio of secreted holoCp to apoCp ( P = 0.038). The role of specific chloride channels was examined by cotransfection of ceruloplasmin and the chloride channel. Overexpression of ClC-4 doubled copper incorporation into ceruloplasmin ( P = 0.011), whereas identical overexpression of ClC-3 had no effect. The effect of ClC-4 was most pronounced under copper-limiting conditions in which it increased copper incorporation more than 4-fold ( P = 0.037). ClC-4 protein was abundant in hepatocyte membranes and was localized in intracellular vesicles containing ATP7B. Conclusions: ClC-4 is an intracellular chloride channel that stimulates copper incorporation into ceruloplasmin, probably by improving the efficiency of the ATP7B copper pump. It is thus an important component of the regulation of hepatic copper transport and may modulate Cu transport rates during copper deficiency, Wilson’s disease, and other copper toxicosis syndromes.