Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Nature volume 453, pages 788–792 (2008)Cite this article

Abstract

Lysosomes are the stomachs of the cell—terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport1,2. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis3. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels4, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5)5,6. However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: 36 Cl - flux in native rat liver lysosomes.
Figure 2: Fluorescence monitoring of lysosomal H + and Cl - flux.
Figure 3: ClC-7 mediates lysosomal Cl - /H + antiport.
Figure 4: ClC-7 is essential for lysosomal acidification in vivo.

References

  1. Dell’Antone, P. Evidence for an ATP-driven ‘proton pump’ in rat liver lysosomes by basic dyes uptake. Biochem. Biophys. Res. Commun. 86, 180–189 (1979)

    Article  Google Scholar 

  2. Ohkuma, S., Moriyama, Y. & Takano, T. Identification and characterization of a proton pump on lysosomes by fluorescein-isothiocyanate-dextran fluorescence. Proc. Natl Acad. Sci. USA 79, 2758–2762 (1982)

    Article  ADS  CAS  Google Scholar 

  3. Kornak, U. et al. Loss of the ClC-7 chloride channel leads to osteopetrosis in mice and man. Cell 104, 205–215 (2001)

    Article  CAS  Google Scholar 

  4. White, M. M. & Miller, C. A voltage-gated anion channel from the electric organ of Torpedo californica . J. Biol. Chem. 254, 10161–10166 (1979)

    CAS  PubMed  Google Scholar 

  5. Picollo, A. & Pusch, M. Chloride/proton antiporter activity of mammalian CLC proteins ClC-4 and ClC-5. Nature 436, 420–423 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Scheel, O., Zdebik, A. A., Lourdel, S. & Jentsch, T. J. Voltage-dependent electrogenic chloride/proton exchange by endosomal CLC proteins. Nature 436, 424–427 (2005)

    Article  ADS  CAS  Google Scholar 

  7. Graham, J. M. in Current Protocols in Cell Biology (eds Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. & Yamada, K. M.) 3.6.1–3.6.21 (Wiley, Hoboken, NJ, 2000)

    Google Scholar 

  8. Goldberg, A. F. & Miller, C. Solubilization and functional reconstitution of a chloride channel from Torpedo californica electroplax. J. Membr. Biol. 124, 199–206 (1991)

    Article  CAS  Google Scholar 

  9. Garty, H., Rudy, B. & Karlish, S. J. A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles. J. Biol. Chem. 258, 13094–13099 (1983)

    CAS  PubMed  Google Scholar 

  10. Rychkov, G. Y., Pusch, M., Roberts, M. L., Jentsch, T. J. & Bretag, A. H. Permeation and block of the skeletal muscle chloride channel, ClC-1, by foreign anions. J. Gen. Physiol. 111, 653–665 (1998)

    Article  CAS  Google Scholar 

  11. Jentsch, T. J., Friedrich, T., Schriever, A. & Yamada, H. The CLC chloride channel family. Pflugers Arch. 437, 783–795 (1999)

    Article  CAS  Google Scholar 

  12. Maduke, M., Pheasant, D. J. & Miller, C. High-level expression, functional reconstitution, and quaternary structure of a prokaryotic ClC-type chloride channel. J. Gen. Physiol. 114, 713–722 (1999)

    Article  CAS  Google Scholar 

  13. Accardi, A. & Miller, C. Secondary active transport mediated by a prokaryotic homologue of ClC Cl- channels. Nature 427, 803–807 (2004)

    Article  ADS  CAS  Google Scholar 

  14. Illsley, N. P. & Verkman, A. S. Membrane chloride transport measured using a chloride-sensitive fluorescent probe. Biochemistry 26, 1215–1219 (1987)

    Article  CAS  Google Scholar 

  15. Henriksen, K. et al. Characterization of osteoclasts from patients harboring a G215R mutation in ClC-7 causing autosomal dominant osteopetrosis type II. Am. J. Pathol. 164, 1537–1545 (2004)

    Article  CAS  Google Scholar 

  16. Letizia, C. et al. Type II benign osteopetrosis (Albers-Schonberg disease) caused by a novel mutation in CLCN7 presenting with unusual clinical manifestations. Calcif. Tissue Int. 74, 42–46 (2004)

    Article  CAS  Google Scholar 

  17. Kornak, U., Ostertag, A., Branger, S., Benichou, O. & de Vernejoul, M. C. Polymorphisms in the CLCN7 gene modulate bone density in postmenopausal women and in patients with autosomal dominant osteopetrosis type II. J. Clin. Endocrinol. Metab. 91, 995–1000 (2006)

    Article  CAS  Google Scholar 

  18. Kasper, D. et al. Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration. EMBO J. 24, 1079–1091 (2005)

    Article  CAS  Google Scholar 

  19. De Angeli, A. et al. The nitrate/proton antiporter AtCLCa mediates nitrate accumulation in plant vacuoles. Nature 442, 939–942 (2006)

    Article  ADS  CAS  Google Scholar 

  20. Jentsch, T. J. Chloride and the endosomal–lysosomal pathway: emerging roles of CLC chloride transporters. J. Physiol. (Lond.) 578, 633–640 (2007)

    Article  CAS  Google Scholar 

  21. Schaller, S. et al. The chloride channel inhibitor NS3736 prevents bone resorption in ovariectomized rats without changing bone formation. J. Bone Miner. Res. 19, 1144–1153 (2004)

    Article  CAS  Google Scholar 

  22. Gottlieb, R. A. & Adachi, S. Nitrogen cavitation for cell disruption to obtain mitochondria from cultured cells. Methods Enzymol. 322, 213–221 (2000)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank K. Swartz, M. Maduke, J. Diamond and R. Youle for critical readings of the manuscript; R. Brady, G. Murray and R. Puertollano-Moro for advice on lysosomes; and the members of the Mindell laboratory for discussions. This work was supported by the NINDS intramural program.

Author information

Authors and Affiliations

  1. Membrane Transport Biophysics Unit, and,,

    Austin R. Graves, Patricia K. Curran & Joseph A. Mindell

  2. Light Microscopy Facility, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 35 Convent Drive, Building 35, MSC 3701, Bethesda, Maryland 20892, USA ,

    Carolyn L. Smith

Authors
  1. Austin R. Graves
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Patricia K. Curran
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Carolyn L. Smith
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Joseph A. Mindell
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joseph A. Mindell.

Supplementary information

Supplementary information

The file contains Supplemental Results, Supplementary Figures 1-5 with Legends and Supplementary Methods. (PDF 815 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Graves, A., Curran, P., Smith, C. et al. The Cl-/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453, 788–792 (2008). https://doi.org/10.1038/nature06907

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature06907

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing