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:

Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters

Nature Structural & Molecular Biology volume 16, pages 1294–1301 (2009)Cite this article

Abstract

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H+-Cl− exchanger of the CLC family of channels and transporters. Cl− affinity depends on the conformation of the protein: it is highest with the extracellular gate removed and weakens as the transporter adopts the occluded configuration and with the intracellular gate removed. The central ion-binding site determines selectivity in CLC transporters and channels. A serine-to-proline substitution at this site confers NO3− selectivity upon the Cl−-specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates through an affinity-switch mechanism and that the bases of substrate specificity are conserved in the CLC channels and transporters.

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: Crystal structure of CLC-ec1.
Figure 2: Cl− binding to WT and mutant CLC-ec1.
Figure 3: Cl− binding to the E148A mutant (PDB 1OTT).
Figure 4: 36Cl binding measured with equilibrium dialysis.
Figure 5: Binding selectivity of WT CLC-ec1 and the E148A mutant.
Figure 6: S107 regulates selectivity.
Figure 7: Chloride dependence of the transport rate.
Figure 8: Comparison of the measured transport rates (black bars) with the theoretical maximal dissociation rate, koffmax, (gray bars) derived from koffmax = Kd*kondiff.

Similar content being viewed by others

References

  1. Price, W.S. & Kuchel, P.W.C.B.A. A 35Cl and 37Cl NMR study of chloride binding to the erythrocyte anion transport protein. Biophys. Chem. 40, 329–337 (1991).

    Article  CAS  Google Scholar 

  2. Fang, Y., Kolmakova-Partensky, L. & Miller, C. A bacterial arginine-agmatine exchange transporter involved in extreme acid resistance. J. Biol. Chem. 282, 176–182 (2007).

    Article  CAS  Google Scholar 

  3. Lockless, S.W., Zhou, M. & MacKinnon, R. Structural and thermodynamic properties of selective ion binding in a K+ channel. PLoS Biol. 5, e121 (2007).

    Article  Google Scholar 

  4. Boudker, O., Ryan, R., Yernool, D., Shimamoto, K. & Gouaux, E. Coupling substrate and ion binding to extracellular gate of a sodium-dependent aspartate transporter. Nature 445, 387–933 (2007).

    Article  CAS  Google Scholar 

  5. Nie, Y., Smirnova, I., Kasho, V. & Kaback, H.R. Energetics of ligand-induced conformational flexibility in the lactose permease of Escherichia coli. J. Biol. Chem. 281, 35779–35984 (2006).

    Article  CAS  Google Scholar 

  6. Singh, S., Yamashita, A. & Gouaux, E. Antidepressant binding site in a bacterial homologue of neurotransmitter transporters. Nature 448, 952–956 (2007).

    Article  CAS  Google Scholar 

  7. Singh, S., Piscitelli, C., Yamashita, A. & Gouaux, E. A competitive inhibitor traps LeuT in an open-to-out conformation. Science 322, 1655–1661 (2008).

    Article  CAS  Google Scholar 

  8. Hille, B. Ion Channels of Excitable Membranes (Sinauer, Sunderland, Massachusetts, USA, 2001).

  9. DiFrancesco, D. & Tortora, P. Direct activation of cardiac pacemaker channels by intracellular cyclic AMP. Nature 351, 145–147 (1991).

    Article  CAS  Google Scholar 

  10. Magleby, K. Gating mechanism of BK (Slo1) channels: so near, yet so far. J. Gen. Physiol. 121, 81–96 (2003).

    Article  CAS  Google Scholar 

  11. Nimigean, C., Shane, T. & Miller, C. A cyclic nucleotide modulated prokaryotic K+ channel. J. Gen. Physiol. 124, 203–210 (2004).

    Article  CAS  Google Scholar 

  12. 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 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. 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  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Graves, A., Curran, P., Smith, C. & Mindell, J. The Cl−/H+ antiporter ClC-7 is the primary chloride permeation pathway in lysosomes. Nature 453, 788–792 (2008).

    Article  CAS  Google Scholar 

  19. Jentsch, T.J. CLC chloride channels and transporters: from genes to protein structure, pathology and physiology. Crit. Rev. Biochem. Mol. Biol. 43, 3–36 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Lobet, S. & Dutzler, R. Ion-binding properties of the ClC chloride selectivity filter. EMBO J. 25, 24–33 (2005).

    Article  Google Scholar 

  22. Accardi, A., Lobet, S., Williams, C., Miller, C. & Dutzler, R. Synergism between halide binding and proton transport in a CLC-type exchanger. J. Mol. Biol. 362, 691–699 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Nguitragool, W. & Miller, C. Uncoupling of a CLC Cl−/H+ exchange transporter by polyatomic anions. J. Mol. Biol. 362, 682–690 (2006).

    Article  CAS  Google Scholar 

  25. Nguitragool, W. & Miller, C. CLC Cl−/H+ transporters constrained by covalent cross-linking. Proc. Natl. Acad. Sci. USA 104, 20659–20665 (2007).

    Article  CAS  Google Scholar 

  26. Walden, M. et al. Uncoupling and turnover in a Cl−/H+ exchange transporter. J. Gen. Physiol. 129, 317–329 (2007).

    Article  CAS  Google Scholar 

  27. Jayaram, H., Accardi, A., Wu, F., Williams, C. & Miller, C. Ion permeation through a Cl−-selective channel designed from a CLC Cl−/H+ exchanger. Proc. Natl. Acad. Sci. USA 105, 11194–11199 (2008).

    Article  CAS  Google Scholar 

  28. Ladbury, J.E. & Doyle, M.L. Biocalorimetry 2: Applications of Calorimetry in the Biological Sciences (Wiley, Hoboken, New Jersey, USA, 2004).

  29. Wiseman, T., Williston, S., Brandts, J.F. & Lin, L.N. Rapid measurement of binding constants and heats of binding using a new titration calorimeter. Anal. Biochem. 179, 131–137 (1989).

    Article  CAS  Google Scholar 

  30. Turnbull, W.B. & Daranas, A.H. On the value of c: can low affinity systems be studied by isothermal titration calorimetry? J. Am. Chem. Soc. 125, 14859–14866 (2003).

    Article  CAS  Google Scholar 

  31. Tellinghuisen, J. Isothermal titration calorimetry at very low c. Anal. Biochem. 373, 395–397 (2008).

    Article  CAS  Google Scholar 

  32. Iyer, R., Iverson, T.M., Accardi, A. & Miller, C. A biological role for prokaryotic ClC chloride channels. Nature 419, 715–718 (2002).

    Article  CAS  Google Scholar 

  33. Accardi, A., Kolmakova-Partensky, L., Williams, C. & Miller, C. Ionic currents mediated by a prokaryotic homologue of CLC Cl− channels. J. Gen. Physiol. 123, 109–119 (2004).

    Article  CAS  Google Scholar 

  34. Lim, H.H. & Miller, C. Intracellular proton-transfer mutants in a CLC Cl−/H+ exchanger. J. Gen. Physiol. 133, 131–138 (2009).

    Article  CAS  Google Scholar 

  35. Faraldo-Gomez, J.D. & Roux, B. Electrostatics of ion stabilization in a ClC chloride channel homologue from Escherichia coli. J. Mol. Biol. 339, 981–1000 (2004).

    Article  CAS  Google Scholar 

  36. Nimigean, C. & Pagel, M. Ligand binding and activation in a prokaryotic cyclic nucleotide-modulated channel. J. Mol. Biol. 371, 1325–1337 (2007).

    Article  CAS  Google Scholar 

  37. Zifarelli, G. & Pusch, M. Conversion of the 2 Cl−/1 H+ antiporter ClC-5 in a NO3−/H+ antiporter by a single point mutation. EMBO J. advance online publication, doi:10.1038/emboj.2008.284 (8 January 2009).

    Article  CAS  Google Scholar 

  38. Ludewig, U., Pusch, M. & Jentsch, T.J. Two physically distinct pores in the dimeric ClC-0 chloride channel. Nature 383, 340–343 (1996).

    Article  CAS  Google Scholar 

  39. Pusch, M., Ludewig, U., Rehfeldt, A. & Jentsch, T.J. Gating of the voltage-dependent chloride channel CIC-0 by the permeant anion. Nature 373, 527–531 (1995).

    Article  CAS  Google Scholar 

  40. 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 

  41. Traverso, S., Elia, L. & Pusch, M. Gating competence of constitutively open CLC-0 mutants revealed by the interaction with a small organic Inhibitor. J. Gen. Physiol. 122, 295–306 (2003).

    Article  CAS  Google Scholar 

  42. Miller, C. & Nguitragool, W. A provisional transport mechanism for a chloride channel-type Cl−/H+ exchanger. Phil. Trans. R. Soc. Lond. B 364, 175–180 (2009).

    Article  CAS  Google Scholar 

  43. Ogawa, H. & Toyoshima, C. Homology modeling of the cation binding sites of Na+K+-ATPase. Proc. Natl. Acad. Sci. USA 99, 15977–15982 (2002).

    Article  CAS  Google Scholar 

  44. Ma, H., Inesi, G. & Toyoshima, C. Substrate-induced conformational fit and headpiece closure in the Ca2+ ATPase (SERCA). J. Biol. Chem. 278, 28938–29043 (2003).

    Article  CAS  Google Scholar 

  45. Inesi, G.M.H., Hua, S. & Toyoshima, C. Characterization of Ca2+ ATPase residues involved in substrate and cation binding. Ann. NY Acad. Sci. 986, 63–71 (2003).

    Article  CAS  Google Scholar 

  46. Toyoshima, C., Nomura, H. & Sugita, Y. Crystal structures of Ca2+-ATPase in various physiological states. Ann. NY Acad. Sci. 986, 1–8 (2003).

    Article  CAS  Google Scholar 

  47. Matulef, K. & Maduke, M. Side-dependent inhibition of a prokaryotic ClC by DIDS. Biophys. J. 89, 1721–1730 (2005).

    Article  CAS  Google Scholar 

  48. Zdebik, A.A. et al. Determinants of anion-proton coupling in mammalian endosomal CLC proteins. J. Biol. Chem. 283, 4219–4227 (2008).

    Article  CAS  Google Scholar 

  49. Richard, H. & Foster, J. Escherichia coli glutamate- and arginine-dependent acid resistance systems increase internal pH and reverse transmembrane potential. J. Bacteriol. 186, 6032–6041 (2004).

    Article  CAS  Google Scholar 

  50. Gut, H. et al. Escherichia coli acid resistance: pH-sensing, activation by chloride and autoinhibition in GadB. EMBO J. 25, 2643–2651 (2006).

    Article  CAS  Google Scholar 

  51. Ludewig, U., Jentsch, T.J. & Pusch, M. Analysis of a protein region involved in permeation and gating of the voltage-gated Torpedo chloride channel ClC-0. J. Physiol. (Lond.) 498, 691–702 (1997).

    Article  CAS  Google Scholar 

  52. Rychkov, G., Pusch, M., Roberts, M. & Bretag, A. Interaction of hydrophobic anions with the rat skeletal muscle chloride channel ClC-1: effects on permeation and gating. J. Physiol. (Lond.) 530, 379–393 (2001).

    Article  CAS  Google Scholar 

  53. Pusch, M., Jordt, S.E., Stein, V. & Jentsch, T.J. Chloride dependence of hyperpolarization-activated chloride channel gates. J. Physiol. (Lond.) 515, 341–353 (1999).

    Article  CAS  Google Scholar 

  54. Bergsdorf, E.Y., Zdebik, A.A. & Jentsch, T.J. Residues important for nitrate/proton coupling in plant and mammalian CLC transporters. J. Biol. Chem. 284, 11184–11193 (2009).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors wish to thank C. Miller (Brandeis University) for unrelenting constructive criticism and for the generous gifts of the CLC-ec1 and CLC-0 clones, K. Campbell, S. England, S. Khademi, R. Subramanian and D. Segaloff for comments on the manuscript, J. Lueck for helpful discussions and comments on the manuscript, C. Blaumueller for expert editing and C. Hills for technical assistance. This work was supported by grant 1R01GM085232 from the US National Institutes of Health to A.A.

Author information

Authors and Affiliations

  1. Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Alessandra Picollo, Mattia Malvezzi & Alessio Accardi

  2. Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa, USA

    Jon C D Houtman

Authors
  1. Alessandra Picollo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mattia Malvezzi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jon C D Houtman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alessio Accardi
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.A. designed research; A.P., M.M. and A.A. performed experiments; A.P., M.M., J.H. and A.A. analyzed the data; A.P. and A.A. wrote the paper.

Corresponding author

Correspondence to Alessio Accardi.

Supplementary information

Supplementary Text and Figures

Supplementary Table 1, Supplementary Figures 1–7 and Supplementary Methods (or Discussion or Data) (PDF 4016 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Picollo, A., Malvezzi, M., Houtman, J. et al. Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters. Nat Struct Mol Biol 16, 1294–1301 (2009). https://doi.org/10.1038/nsmb.1704

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1704

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