The need for proton-driven vesicular Cl- accumulation: insights from human disease, designer mice and biophysics

Five mammalian members of the CLC family of chloride channels and transporters, ClC-3 through ClC-7, are predominantly expressed on vesicles of the endoso­mal-lysosomal system. They were previously thought to be chloride channels (like ClC-1, -2, -Ka and –Kb) and their role was seen in providing countercurrents for electrogenic vesicular proton pumps. Without such neutralizing currents these H⁺-ATPases would not be able to acidify the cell interior. The diverse phenotypes observed upon loss of vesicular CLC proteins (e.g. impaired endocytosis and kidney stones with ClC-5, and osteopetrosis and lysosomal storage disease with ClC-7) were mostly attributed to such acidification defects.

Over the past years it emerged that vesicular CLC proteins are Cl^-/H⁺-exchangers rather than Cl^- channels. Although these electrogenic exchangers can also provide countercurrents for vesicular acidification, the question arose why Nature has preferred Cl^-/H⁺-exchangers over Cl­^- channels when the latter are perfectly suited for the generating countercurrents.

To address this question, we designed ‘unc’ mice in which we converted selected CLC Cl^-/H⁺-exchangers into pure, uncoupled chloride conductances using single point mutations. This can be achieved by neutralizing a key ‘gating glutamate’ in the permeation pathway of CLC exchangers. We generated both ClC-5^unc and ClC-7^unc mice as models for an early endosomal and a lysosomal CLC transporter, respectively. Any phenotype detectable in those knock-in mice cannot be attributed to impaired acidification.

Whereas renal cortical endosomes of ClC-5 KO mice displayed impaired ATP-driven luminal acidification, endosomes from Clcn5^unc mice acidified normally as expected. Surprisingly, however, proximal tubular endocytosis was as severely affected in Clcn5^unc mice as in Clcn5^-- mice. We conclude that ClC-5 is important for the acidification of renal endosomes, but that normal endosomal acidification is not sufficient for renal endocytosis. There is a crucial need for Cl^-/H⁺-exchange, pointing to a role in proton-gradient driven Cl^- accumulation in endocytosis.

Disruption of the late endosomal/lysosomal ClC-7 leads to osteopetrosis and lysosomal storage in men and mice. Clcn7^unc mice show lysosomal storage disease that is as severe as in the KO, but their osteopetrosis is milder. As ClC-7 cannot be expressed at the plasma membrane, we characterized lysosomal H⁺ transport in intact cells and isolated lysosomes to demonstrate that ClC-7 mediates indeed Cl^-/H⁺-exchange and that the unc mutation converts it into an uncoupled anion conductance. We showed that lysosomal pH is unchanged in either Clcn7^-/- or Clcn7^unc/unc mice. In contrast to endosomes, lysosomal acidification does not need a CLC protein and not even anions: lysosomes display a significant cation conductance. Using a dextran-coupled ratioable Cl^--sensitive dye, we showed that lysosomes indeed accumulate chloride. These experiments were backed up by model calculations. Our studies show that proton-driven chloride accumulation is important all along the endosomal-lysosomal pathway and suggest an important, hitherto unknown role of luminal chloride concentration.