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.

  • Article
  • Published:

Large movement in the C terminus of CLC-0 chloride channel during slow gating

Nature Structural & Molecular Biology volume 13, pages 1115–1119 (2006)Cite this article

Abstract

Chloride channels and transporters of the CLC gene family are expressed in virtually all cell types and are crucial in the regulation of membrane potential, chloride homeostasis and intravesicular pH. There are two gating processes that open CLC channels—fast and slow. The fast gating process in CLC channels has recently been linked to a small movement of a glutamate side chain. However, the molecular mechanism underlying the slow gating process is still elusive. Using spectroscopic microscopy, we observed a large backbone movement in the C terminus of the CLC-0 chloride channel that was functionally linked to slow gating. We further showed that the C-terminal movement had a time course similar to slow gating. In addition, a mutation known to lock the slow gate open prevented movement of the C terminus. When combined with recent structural information on the CLC C terminus, our findings provide a structural model for understanding the conformational changes linked to slow gating in CLC transport proteins.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Gating of CLC-0 channels.
Figure 2: Spectra FRET measurements.
Figure 3: Comparison of FRET and current time courses.
Figure 4: FRET changes during slow gating.

Similar content being viewed by others

Accession codes

Accessions

Protein Data Bank

References

  1. Jentsch, T.J., Steinmeyer, K. & Schwarz, G. Primary structure of Torpedo marmorata chloride channel isolated by expression cloning in Xenopus oocytes. Nature 348, 510–514 (1990).

    Article  CAS  Google Scholar 

  2. Chen, T.Y. Structure and function of clc channels. Annu. Rev. Physiol. 67, 809–839 (2005).

    Article  CAS  Google Scholar 

  3. Jentsch, T.J., Neagoe, I. & Scheel, O. CLC chloride channels and transporters. Curr. Opin. Neurobiol. 15, 319–325 (2005).

    Article  CAS  Google Scholar 

  4. Miller, C. ClC chloride channels viewed through a transporter lens. Nature 440, 484–489 (2006).

    Article  CAS  Google Scholar 

  5. Pusch, M. & Jentsch, T.J. Unique structure and function of chloride transporting CLC proteins. IEEE Trans. Nanobioscience 4, 49–57 (2005).

    Article  Google Scholar 

  6. Dutzler, R., Campbell, E.B. & MacKinnon, R. Gating the selectivity filter in ClC chloride channels. Science 300, 108–112 (2003).

    Article  CAS  Google Scholar 

  7. Accardi, A. et al. Separate ion pathways in a Cl−/H+ exchanger. J. Gen. Physiol. 126, 563–570 (2005).

    Article  CAS  Google Scholar 

  8. Miller, C. & White, M.M. A voltage-dependent chloride conductance channel from Torpedo electroplax membrane. Ann. NY Acad. Sci. 341, 534–551 (1980).

    Article  CAS  Google Scholar 

  9. Chen, T.Y. & Miller, C. Nonequilibrium gating and voltage dependence of the ClC-0 Cl− channel. J. Gen. Physiol. 108, 237–250 (1996).

    Article  CAS  Google Scholar 

  10. Fong, P., Rehfeldt, A. & Jentsch, T.J. Determinants of slow gating in ClC-0, the voltage-gated chloride channel of Torpedo marmorata. Am. J. Physiol. 274, C966–C973 (1998).

    Article  CAS  Google Scholar 

  11. Estevez, R., Pusch, M., Ferrer-Costa, C., Orozco, M. & Jentsch, T.J. Functional and structural conservation of CBS domains from CLC chloride channels. J. Physiol. (Lond.) 557, 363–378 (2004).

    Article  CAS  Google Scholar 

  12. Hebeisen, S. et al. The role of the carboxyl terminus in ClC chloride channel function. J. Biol. Chem. 279, 13140–13147 (2004).

    Article  CAS  Google Scholar 

  13. Glauner, K.S., Mannuzzu, L.M., Gandhi, C.S. & Isacoff, E.Y. Spectroscopic mapping of voltage sensor movement in the Shaker potassium channel. Nature 402, 813–817 (1999).

    Article  CAS  Google Scholar 

  14. Cha, A., Snyder, G.E., Selvin, P.R. & Bezanilla, F. Atomic scale movement of the voltage-sensing region in a potassium channel measured via spectroscopy. Nature 402, 809–813 (1999).

    Article  CAS  Google Scholar 

  15. Posson, D.J., Ge, P., Miller, C., Bezanilla, F. & Selvin, P.R. Small vertical movement of a K+ channel voltage sensor measured with luminescence energy transfer. Nature 436, 848–851 (2005).

    Article  CAS  Google Scholar 

  16. Chanda, B., Asamoah, O.K., Blunck, R., Roux, B. & Bezanilla, F. Gating charge displacement in voltage-gated ion channels involves limited transmembrane movement. Nature 436, 852–856 (2005).

    Article  CAS  Google Scholar 

  17. Lakowicz, J. Principles of Fluorescence Spectroscopy 725 (Plenum, New York, 1999).

    Book  Google Scholar 

  18. Selvin, P.R. The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734 (2000).

    Article  CAS  Google Scholar 

  19. Stryer, L. & Haugland, R.P. Energy transfer: a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

    Article  CAS  Google Scholar 

  20. Stryer, L. Fluorescence energy transfer as a spectroscopic ruler. Annu. Rev. Biochem. 47, 819–846 (1978).

    Article  CAS  Google Scholar 

  21. Rizzo, M.A., Springer, G.H., Granada, B. & Piston, D.W. An improved cyan fluorescent protein variant useful for FRET. Nat. Biotechnol. 22, 445–449 (2004).

    Article  CAS  Google Scholar 

  22. Heim, R. & Tsien, R.Y. Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr. Biol. 6, 178–182 (1996).

    Article  CAS  Google Scholar 

  23. Takanishi, C.L., Bykova, E., Cheng, W. & Zheng, J. Quantification of GFP-based FRET in transfected cells. Brain Res. 1091, 132–139 (2006).

    Article  CAS  Google Scholar 

  24. Zheng, J. Spectroscopy-based quantitative fluorescence resonance energy transfer analysis. Methods Mol. Biol. 337, 65–78 (2006).

    CAS  PubMed  Google Scholar 

  25. Zheng, J., Trudeau, M.C. & Zagotta, W.N. Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit. Neuron 36, 891–896 (2002).

    Article  CAS  Google Scholar 

  26. Dutzler, R., Campbell, E.B., Cadene, M., Chait, B.T. & MacKinnon, R. X-ray structure of a ClC chloride channel at 3.0 Å reveals the molecular basis of anion selectivity. Nature 415, 287–294 (2002).

    Article  CAS  Google Scholar 

  27. Meyer, S. & Dutzler, R. Crystal structure of the cytoplasmic domain of the chloride channel ClC-0. Structure 14, 299–307 (2006).

    Article  CAS  Google Scholar 

  28. Chen, T.Y. Extracellular zinc ion inhibits ClC-0 chloride channels by facilitating slow gating. J. Gen. Physiol. 112, 715–726 (1998).

    Article  CAS  Google Scholar 

  29. Lin, Y.W., Lin, C.W. & Chen, T.Y. Elimination of the slow gating of ClC-0 chloride channel by a point mutation. J. Gen. Physiol. 114, 1–12 (1999).

    Article  CAS  Google Scholar 

  30. Miller, C. & White, M.M. Dimeric structure of single chloride channels from Torpedo electroplax. Proc. Natl. Acad. Sci. USA 81, 2772–2775 (1984).

    Article  CAS  Google Scholar 

  31. Pusch, M., Ludewig, U. & Jentsch, T.J. Temperature dependence of fast and slow gating relaxations of ClC-0 chloride channels. J. Gen. Physiol. 109, 105–116 (1997).

    Article  CAS  Google Scholar 

  32. Bennetts, B., Roberts, M.L., Bretag, A.H. & Rychkov, G.Y. Temperature dependence of human muscle ClC-1 chloride channel. J. Physiol. (Lond.) 535, 83–93 (2001).

    Article  CAS  Google Scholar 

  33. Jordt, S.E., McKemy, D.D. & Julius, D. Lessons from peppers and peppermint: the molecular logic of thermosensation. Curr. Opin. Neurobiol. 13, 487–492 (2003).

    Article  CAS  Google Scholar 

  34. Zagotta, W.N. et al. Structural basis for modulation and agonist specificity of HCN pacemaker channels. Nature 425, 200–205 (2003).

    Article  CAS  Google Scholar 

  35. Simon, D.B. et al. Mutations in the chloride channel gene, CLCNKB, cause Bartter's syndrome type III. Nat. Genet. 17, 171–178 (1997).

    Article  CAS  Google Scholar 

  36. Haug, K. et al. Mutations in CLCN2 encoding a voltage-gated chloride channel are associated with idiopathic generalized epilepsies. Nat. Genet. 33, 527–532 (2003).

    Article  CAS  Google Scholar 

  37. Steinmeyer, K. et al. Inactivation of muscle chloride channel by transposon insertion in myotonic mice. Nature 354, 304–308 (1991).

    Article  CAS  Google Scholar 

  38. Lloyd, S.E. et al. A common molecular basis for three inherited kidney stone diseases. Nature 379, 445–449 (1996).

    Article  CAS  Google Scholar 

  39. He, L., Denton, J., Nehrke, K. & Strange, K. Carboxy terminus splice variation alters ClC channel gating and extracellular cysteine reactivity. Biophys. J. 90, 3570–3581 (2006).

    Article  CAS  Google Scholar 

  40. Schmidt-Rose, T. & Jentsch, T.J. Reconstitution of functional voltage-gated chloride channels from complementary fragments of CLC-1. J. Biol. Chem. 272, 20515–20521 (1997).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to D. Piston (Vanderbilt University), R. Tsien (University of California at San Diego) and I. Kojima (Gunma University) for the kind gifts of Cerulean, EYFP and mTRPV2, respectively. We also thank E. Lachica (Olympus USA) for support of the Spectra FRET imaging system and W. Cheng, C. Takanishi and W.-P. Yu for technical assistance. This work was supported by grants from the American Heart Association (0665201Y to J.Z.) and the US National Institutes of Health (R01-GM065447 to T.-Y.C.).

Author information

Authors and Affiliations

  1. Department of Physiology and Membrane Biology, University of California, Davis, 95616, California, USA

    Ekaterina A Bykova & Jie Zheng

  2. Center for Neuroscience, University of California, Davis, 95616, California, USA

    Xiao-Dong Zhang & Tsung-Yu Chen

  3. Department of Neurology, University of California, Davis, 95616, California, USA

    Tsung-Yu Chen

Authors
  1. Ekaterina A Bykova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao-Dong Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Tsung-Yu Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jie Zheng
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jie Zheng.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bykova, E., Zhang, XD., Chen, TY. et al. Large movement in the C terminus of CLC-0 chloride channel during slow gating. Nat Struct Mol Biol 13, 1115–1119 (2006). https://doi.org/10.1038/nsmb1176

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1176

This article is cited by

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