ClC-3 chloride channels facilitate endosomal acidification and chloride accumulation

We investigated the involvement of ClC-3 chloride channels in endosomal acidification by measurement of endosomal pH and chloride concentration [Cl ^-] in control versus ClC-3-deficient hepatocytes and in control versus ClC-3-transfected Chinese hamster ovary cells. Endosomes were labeled with pH or [Cl^-]-sensing fluorescent transferrin (Tf), which targets to early/recycling endosomes, or α₂-macroglobulin (α₂M), which targets to late endosomes. In pulse label-chase experiments, [Cl^-] was 19 mM just after internalization in α₂M-labeled endosomes in primary cultures of hepatocytes from wild-type mice, increasing to 58 nM over 45 min, whereas pH decreased from 7.1 to 5.4. Endosomal acidification and [Cl^-] accumulation were significantly impaired in hepatocytes from ClC-3 knock-out mice, with [Cl ^-] increasing from 16 to 43 mM and pH decreasing from 7.1 to 6.0. Acidification and Cl^- accumulation were blocked by bafilomycin. In Tf-labeled endosomes, [Cl^-] was 46 mM in wild-type versus 35 mM in ClC-3-deficient hepatocytes at 15 min after internalization, with corresponding pH of 6.1 versus 6.5. Approximately 4-fold increased Cl^- conductance was found in α₂M-labeled endosomes isolated from hepatocytes of wild-type versus ClC-3 null mice. In contrast, Golgi acidification was not impaired in ClC-3-deficient hepatocytes. In transfected Chinese hamster ovary cells expressing ClC-3A, endosomal acidification and [Cl^-] accumulation were enhanced. [Cl^-] in α₂M-labeled endosomes was 42 mM (control) versus 53 mM (ClC-3A) at 45 min, with corresponding pH 5.8 versus 5.2; [Cl^-] in Tf-labeled endosomes at 15 min was 37 mM (control) versus 49 mM (ClC-3A) with pH 6.3 versus 5.9. Our results provide direct evidence for involvement of ClC-3 in endosomal acidification by Cl^- shunting of the interior-positive membrane potential created by the vacuolar H⁺ pump.