Mutations in the ClC-7/Ostm1 ion transporter lead to osteopetrosis and lysosomal storage disease. Its lysosomal localization hitherto precluded detailed functional characterization. Using a mutated ClC-7 that reaches the plasma membrane, we now show that both the aminoterminus and transmembrane span of the Ostm1 b-subunit are required for ClC-7 Cl À /H þ -exchange, whereas the Ostm1 transmembrane domain suffices for its ClC-7-dependent trafficking to lysosomes. ClC-7/Ostm1 currents were strongly outwardly rectifying owing to slow gating of ion exchange, which itself displays an intrinsically almost linear voltage dependence. Reversal potentials of tail currents revealed a 2ClSeveral disease-causing CLCN7 mutations accelerated gating. Such mutations cluster to the second cytosolic cystathionine-b-synthase domain and potential contact sites at the transmembrane segment. Our work suggests that gating underlies the rectification of all endosomal/ lysosomal CLCs and extends the concept of voltage gating beyond channels to ion exchangers.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl À concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl À /H + exchange, we now generate Clcn3 unc/unc mice in which ClC-3 is converted into a Cl À channel. Unlike Clcn3 À/À mice, Clcn3 unc/unc mice appear normal owing to compensation by ClC-4 with which ClC-3 forms heteromers. ClC-4 protein levels are strongly reduced in Clcn3 À/À , but not in Clcn3 unc/unc mice because ClC-3 unc binds and stabilizes ClC-4 like wild-type ClC-3. Although mice lacking ClC-4 appear healthy, its absence in Clcn3 unc/unc /Clcn4 À/À mice entails even stronger neurodegeneration than observed in Clcn3 À/À mice. A fraction of ClC-3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3 unc/unc or Clcn3 À/À mice before neurodegeneration sets in. Both, Cl À /H + -exchange activity and the stabilizing effect on ClC-4, are central to the biological function of ClC-3.

Background: ClC-7 is a homodimeric lysosomal chloride transporter important for lysosomal function and bone degradation. Results: Altered gating kinetics of one subunit affect the kinetics of the other subunit. Conclusion: Gating of ClC-7 involves both CLC subunits and requires noncovalent binding of cytoplasmic domains. Significance: Osteopetrosis and lysosomal storage disease are associated with accelerating mutations in the ClC-7 C terminus and the contacting intramembrane part.

Chloride-proton exchange by the lysosomal anion transporter ClC-7/Ostm1 is of pivotal importance for the physiology of lysosomes and bone resorption. Mice lacking either ClC-7 or Ostm1 develop a lysosomal storage disease and mutations in either protein have been found to underlie osteopetrosis in mice and humans. Some human disease-causing CLCN7 mutations accelerate the usually slow voltage-dependent gating of ClC-7/Ostm1. However, it has remained unclear whether the fastened kinetics is indeed causative for the disease. Here we identified and characterized a new deleterious ClC-7 mutation in Belgian Blue cattle with a severe symptomatology including perinatal lethality and in most cases gingival hamartomas. By autozygosity mapping and genome-wide sequencing we found a handful of candidate variants, including a cluster of three private SNPs causing the substitution of a conserved tyrosine in the CBS2 domain of ClC-7 by glutamine. The case for ClC-7 was strengthened by subsequent examination of affected calves that revealed severe osteopetrosis. The Y750Q mutation largely preserved the lysosomal localization and assembly of ClC-7/Ostm1, but drastically accelerated its activation by membrane depolarization. These data provide first evidence that accelerated ClC-7/Ostm1 gating per se is deleterious, highlighting a physiological importance of the slow voltage-activation of ClC-7/Ostm1 in lysosomal function and bone resorption.