Publications

• The late endosomal ClC-6 mediates proton/chloride countertransport in heterologous plasma membrane expression.

  Neagoe I., Stauber T., Fidzinski P., Bergsdorf E.Y., Jentsch T.J.

  Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these ... >> More

  Members of the CLC protein family of Cl(-) channels and transporters display the remarkable ability to function as either chloride channels or Cl(-)/H(+) antiporters. Due to the intracellular localization of ClC-6 and ClC-7, it has not yet been possible to study the biophysical properties of these members of the late endosomal/lysosomal CLC branch in heterologous expression. Whereas recent data suggest that ClC-7 functions as an antiporter, transport characteristics of ClC-6 have remained entirely unknown. Here, we report that fusing the green fluorescent protein (GFP) to the N terminus of ClC-6 increased its cell surface expression, allowing us to functionally characterize ClC-6. Compatible with ClC-6 mediating Cl(-)/H(+) exchange, Xenopus oocytes expressing GFP-tagged ClC-6 alkalinized upon depolarization. This alkalinization was dependent on the presence of extracellular anions and could occur against an electrochemical proton gradient. As observed in other CLC exchangers, ClC-6-mediated H(+) transport was abolished by mutations in either the "gating" or "proton" glutamate. Overexpression of GFP-tagged ClC-6 in CHO cells elicited small, outwardly rectifying currents with a Cl(-) > I(-) conductance sequence. Mutating the gating glutamate of ClC-6 yielded an ohmic anion conductance that was increased by additionally mutating the "anion-coordinating" tyrosine. Additionally changing the chloride-coordinating serine 157 to proline increased the NO(3)(-) conductance of this mutant. Taken together, these data demonstrate for the first time that ClC-6 is a Cl(-)/H(+) antiporter. << Less

  J. Biol. Chem. 285:21689-21697(2010) [PubMed] [EuropePMC]

• A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl-/H+-Exchanger, Causes Early-Onset Neurodegeneration.

  Polovitskaya M.M., Barbini C., Martinelli D., Harms F.L., Cole F.S., Calligari P., Bocchinfuso G., Stella L., Ciolfi A., Niceta M., Rizza T., Shinawi M., Sisco K., Johannsen J., Denecke J., Carrozzo R., Wegner D.J., Kutsche K., Tartaglia M., Jentsch T.J.

  Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembra ... >> More

  Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl^-/H⁺ exchangers function as electric shunts for proton pumping and in luminal Cl^- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl^-/H⁺-exchanger ClC-6. Whereas Clcn6^-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6^Tyr553Cys, but not ClC-6^WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl^-/H⁺ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6^{Tyr553Cys/Glu200Ala} or ClC-6^Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H⁺-driven Cl^- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function. << Less

  Am. J. Hum. Genet. 107:1062-1077(2020) [PubMed] [EuropePMC]