Involvement of chloride channels in hepatic copper metabolism: ClC-4 promotes copper incorporation into ceruloplasmin  Ting Wang, Steven A. Weinman  Gastroenterology  Volume 126, Issue 4, Pages 1157-1166 (April 2004) DOI: 10.1053/j.gastro.2004.01.015

Figure 1 Effect of chloride removal on holoceruloplasmin secretion. Culture medium from CHO-K1 cells was collected at 12-hour intervals and subjected to native PAGE and immunoblotting for ceruloplasmin. (A) Native-PAGE immunoblot of holoceruloplasmin secreted into culture medium by ceruloplasmin-transfected cells cultured in normal, 150 mmol/L, chloride medium (N), or low, 14 mmol/L, chloride medium (L). (B) Densitometry analysis of 3 identical immunoblot experiments for medium collected over the 0- to 12-hour interval after exposure to low chloride (∗P = 0.038). (C) Effect of low-chloride medium on intracellular content of ceruloplasmin and enhanced green fluorescent protein. Cells were transfected with either protein and cultured in either normal or low-chloride medium. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 2 Effect of chloride substitution on relative apoceruloplasmin and holoceruloplasmin secretion. Ceruloplasmin-transfected CHO-K1 cells were cultured and medium was collected from the 12- to 24-hour interval. Apoceruloplasmin (ApoCp) and holoCp were resolved by SDS-PAGE and immunoblotting. (A) Effect of addition of CuCl2 (50 μmol/L) or the copper chelator TEPA (50 μmol/L) on the relative apoCp and holoCp secretion. (B) ApoCp and holoCp were measured in medium collected from cells cultured in either normal or low-chloride culture medium. (C) Summary of densitometry analysis of 3 identical immunoblot experiments as in B (∗P = 0.03). Values are expressed as 100 × holoCp/(holoCp + apoCp). Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 3 Effect of chloride substitution on ceruloplasmin secretion in hepatoma cells. Huh-7 hepatoma cells were cultured in either normal (N) or low- (L) chloride medium as described. Culture medium was collected over the 12- to 24-hour interval and subjected to SDS-PAGE immunoblot analysis. (A) Example of relative proportion of holoCp and apoCp in the medium. (B) Summary of densitometry analysis (n = 3, ∗P = 0.036). Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 4 Lack of effect of low chloride on ATP7B expression or distribution. Huh-7 cells were cultured in normal or low-chloride medium for 24 hours and examined for presence of ATP7B. (A) Immunofluorescence of ATP7B in cells cultured in normal-chloride medium. (B) Immunofluorescence of ATP7B in cells cultured in low-chloride medium. (C) Western blot for quantitation of ATP7B expression in cell lysates from normal (N) or low- (L) chloride cultured cells. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 5 Expression of ClC chloride channels in CHO-K1 cells. Cell protein was analyzed by SDS-PAGE and immunoblotting for ClC-3 or ClC-4. Lane 1, cell lysate from transiently transfected cells. Lane 2, cell lysate from native CHO-K1 cells. Lane 3, P-100 membrane pellet from native CHO-K1 cells. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 6 Effect of chloride channel overexpression on relative apoceruloplasmin and holoceruloplasmin secretion. CHO-K1 cells were transfected transiently with ceruloplasmin and either control LacZ, ClC-3, or ClC-4 plasmid. Medium was collected 24 hours after transfection and apoCp and holoCp were measured by immunoblotting. (A) Proportion of apoCp and holoCp under control conditions. (B) Western blots of cell lysates showing the expected expression of the indicated chloride channel. (C) Summary of densitometry analysis of multiple experiments as in A. ∗P = 0.011. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 7 Copper dependence of chloride channel effects. Cells were transfected with ceruloplasmin and either control LacZ or ClC-4 plasmid and cultured with either control medium, medium supplemented with 50 μmol/L CuCl2, or medium with 2–4 μmol/L TEPA added. Culture medium was collected over the 0- to 24-hour interval. ApoCp and holoCp were examined by SDS-PAGE. (A) Representative immunoblots showing ceruloplasmin content are presented. (B) ClC-4 expression level was documented by immunoblotting of cell lysates. (C) Summary of densitometry analysis of multiple experiments as in A. ∗P < 0.05. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 8 Expression of ClC channels in mouse liver and other tissues. Expression of chloride channel proteins was examined by immunoblotting of P-100 membrane pellets derived from normal mouse tissues. The total sample applied was 50 μg of protein in each case. (A) Membrane fractions were derived from tissue homogenates and probed as described in the Materials and Methods section. L, liver; B, brain; K, kidney; I, ileum; H, heart; S, spleen; M, skeletal muscle; Lu, lung. (B) Protein levels were compared in P-100 membrane fractions derived from either whole liver tissue (L) or purified hepatocytes (Hep). Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)

Figure 9 Association of ATP7B and ClC-4. (A) Total liver homogenates were incubated with magnetic beads coated with anti-ATP7B antibodies and the resulting precipitates were run on SDS-PAGE and immunoblotted for several different cellular proteins. Lane 1, total liver membrane fraction. Lane 2, anti-ATP7B immunoprecipitate. Lane 3, control IgG immunoprecipitate. (B) Immunoprecipitation was performed identically except using 1% NP-40, 0.2% SDS detergent-treated liver homogenate in lanes 2 and 3. Gastroenterology 2004 126, 1157-1166DOI: (10.1053/j.gastro.2004.01.015)