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A structural link between inactivation and block of a K+ channel

Nature Structural & Molecular Biology volume 15, pages 605–612 (2008)Cite this article

Abstract

Gating the ion-permeation pathway in K+ channels requires conformational changes in activation and inactivation gates. Here we have investigated the structural alterations associated with pH-dependent inactivation gating of the KcsA-Kv1.3 K+ channel using solid-state NMR spectroscopy in direct reference to electrophysiological and pharmacological experiments. Transition of the KcsA-Kv1.3 K+ channel from a closed state at pH 7.5 to an inactivated state at pH 4.0 revealed distinct structural changes within the pore, correlated with activation-gate opening and inactivation-gate closing. In the inactivated K+ channel, the selectivity filter adopts a nonconductive structure that was also induced by binding of a pore-blocking tetraphenylporphyrin derivative. The results establish a structural link between inactivation and block of a K+ channel in a membrane setting.

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Figure 1: Inactivation and KTX binding properties of the KcsA-Kv1.3 channel.
Figure 2: Comparison of ssNMR data for KcsA-Kv1.3 at pH 7.5, pH 4.0, and in the porphyrin-bound state.
Figure 3: Analysis of changes in KcsA-Kv1.3 backbone conformation associated with pH-dependent gating and porphyrin binding.
Figure 4: Analysis of porphyrin binding to KcsA-Kv1.3.
Figure 5: Structural models for the KcsA-Kv1.3 channel at pH 4.0 and with bound porphyrin at pH 7.5.

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References

  1. Hille, B. Ionic Channels of Excitable Membranes 3rd edn. (Sinauer Associates Inc., Sunderland, MA, 2001).

    Google Scholar 

  2. Yellen, G. The moving parts of voltage-gated ion channels. Q. Rev. Biophys. 31, 239–295 (1998).

    Article  CAS  Google Scholar 

  3. Jiang, Y. et al. The open pore conformation of potassium channels. Nature 417, 523–526 (2002).

    Article  CAS  Google Scholar 

  4. Bean, B.P. The action potential in mammalian central neurons. Nat. Rev. Neurosci. 8, 451–465 (2007).

    Article  CAS  Google Scholar 

  5. Starkus, J.G., Kuschel, L., Rayner, M.D. & Heinemann, S.H. Ion conduction through C-type inactivated shaker channels. J. Gen. Physiol. 110, 539–550 (1997).

    Article  CAS  Google Scholar 

  6. Starkus, J.G., Kuschel, L., Rayner, M.D. & Heinemann, S.H. Macroscopic Na+ Currents in the “nonconducting” shaker potassium channel mutant W434F. J. Gen. Physiol. 112, 85–93 (1998).

    Article  CAS  Google Scholar 

  7. Valiyaveetil, F.I., Leonetti, M., Muir, T.W. & MacKinnon, R. Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation. Science 314, 1004–1007 (2006).

    Article  CAS  Google Scholar 

  8. Berneche, S. & Roux, B. A gate in the selectivity filter of potassium channels. Structure 13, 591–600 (2005).

    Article  CAS  Google Scholar 

  9. Cordero-Morales, J.F. et al. Molecular determinants of gating at the potassium-channel selectivity filter. Nat. Struct. Mol. Biol. 13, 311–318 (2006).

    Article  CAS  Google Scholar 

  10. Cordero-Morales, J.F., Cuello, L.G. & Perozo, E. Voltage-dependent gating at the KcsA selectivity filter. Nat. Struct. Mol. Biol. 13, 319–322 (2006).

    Article  CAS  Google Scholar 

  11. Cordero-Morales, J.F. et al. Molecular driving forces determining potassium channel slow inactivation. Nat. Struct. Mol. Biol. 14, 1062–1069 (2007).

    Article  CAS  Google Scholar 

  12. Blunck, R., Cordero-Morales, J.F., Cuello, L.G., Perozo, E. & Bezanilla, F. Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction. J. Gen. Physiol. 128, 569–581 (2006).

    Article  CAS  Google Scholar 

  13. Zhou, Y., Morais-Cabral, J.H., Kaufman, A. & MacKinnon, R. Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0Å resolution. Nature 414, 43–48 (2001).

    Article  CAS  Google Scholar 

  14. Long, S.B., Campbell, E.B. & MacKinnon, R. Crystal structure of a mammalian voltage-dependent shaker family K+ channel. Science 309, 897–903 (2005).

    Article  CAS  Google Scholar 

  15. Long, S.B., Tao, X., Campbell, E.B. & MacKinnon, R. Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007).

    Article  CAS  Google Scholar 

  16. Lange, A. et al. Toxin-induced conformational changes in a potassium channel revealed by solid-state NMR. Nature 440, 959–962 (2006).

    Article  CAS  Google Scholar 

  17. Legros, C. et al. Generating a high affinity scorpion toxin receptor in KcsA-Kv1.3 chimeric potassium channels. J. Biol. Chem. 275, 16918–16924 (2000).

    Article  CAS  Google Scholar 

  18. Gradl, S.N., Felix, J.P., Isacoff, E.Y., Garcia, M.L. & Trauner, D. Protein surface recognition by rational design: nanomolar ligands for potassium channels. J. Am. Chem. Soc. 125, 12668–12669 (2003).

    Article  CAS  Google Scholar 

  19. Yu, L. et al. Nuclear magnetic resonance structural studies of a potassium channel-charybdotoxin complex. Biochemistry 44, 15834–15841 (2005).

    Article  CAS  Google Scholar 

  20. Chill, J.H., Louis, J.M., Miller, C. & Bax, A. NMR study of the tetrameric KcsA potassium channel in detergent micelles. Protein Sci. 15, 684–698 (2006).

    Article  CAS  Google Scholar 

  21. Takeuchi, K., Takahashi, H., Kawano, S. & Shimada, I. Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA. J. Biol. Chem. 282, 15179–15186 (2007).

    Article  CAS  Google Scholar 

  22. Baker, K.A., Tzitzilonis, C., Kwiatkowski, W., Choe, S. & Riek, R. Conformational dynamics of the KcsA potassium channel governs gating properties. Nat. Struct. Mol. Biol. 14, 1089–1095 (2007).

    Article  CAS  Google Scholar 

  23. Chill, J.H., Louis, J.M., Delaglio, F. & Bax, A. Local and global structure of the monomeric subunit of the potassium channel KcsA probed by NMR. Biomembranes 1768, 3260–3270 (2007).

    Article  CAS  Google Scholar 

  24. Schneider, R. et al. Solid-state NMR spectroscopy applied to a chimeric potassium channel in lipid bilayers. J. Am. Chem. Soc. (in the press).

  25. Cortes, D.M., Cuello, L.G. & Perozo, E. Molecular architecture of full-length KcsA: role of cytoplasmic domains in ion permeation and activation gating. J. Gen. Physiol. 117, 165–180 (2001).

    Article  CAS  Google Scholar 

  26. Valiyaveetil, F.I., Zhou, Y. & MacKinnon, R. Lipids in the structure, folding, and function of the KcsA K+ channel. Biochemistry 41, 10771–10777 (2002).

    Article  CAS  Google Scholar 

  27. Matthews, E.E., Zoonens, M. & Engelman, D.M. Dynamic helix interactions in transmembrane signaling. Cell 127, 447–450 (2006).

    Article  CAS  Google Scholar 

  28. Cuello, L.G., Romero, J.G., Cortes, D.M. & Perozo, E. pH-dependent gating in the Streptomyces lividans K+ channel. Biochemistry 37, 3229–3236 (1998).

    Article  CAS  Google Scholar 

  29. Heginbotham, L., LeMasurier, M., Kolmakova-Partensky, L. & Miller, C. Single Streptomyces lividans K+ channels: functional asymmetries and sidedness of proton activation. J. Gen. Physiol. 114, 551–560 (1999).

    Article  CAS  Google Scholar 

  30. Luca, S. et al. Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under Magic Angle Spinning. J. Biomol. NMR 20, 325–331 (2001).

    Article  CAS  Google Scholar 

  31. Neal, S., Nip, A.M., Zhang, H. & Wishart, D.S. Rapid and accurate calculation of protein 1H, 13C and 15N chemical shifts. J. Biomol. NMR 26, 215–240 (2003).

    Article  CAS  Google Scholar 

  32. Brünger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    Article  Google Scholar 

  33. Lange, A., Luca, S. & Baldus, M. Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids. J. Am. Chem. Soc. 124, 9704–9705 (2002).

    Article  CAS  Google Scholar 

  34. 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 

  35. Jiang, Y. et al. X-ray structure of a voltage-dependent K+ channel. Nature 423, 33–41 (2003).

    Article  CAS  Google Scholar 

  36. Bielecki, A., Kolbert, A.C. & Levitt, M.H. Frequency-switched pulse sequences: homonuclear decoupling and dilute spin NMR in solids. Chem. Phys. Lett. 155, 341–346 (1989).

    Article  CAS  Google Scholar 

  37. Dominguez, C., Boelens, R. & Bonvin, A.M.J.J. HADDOCK: a protein-protein docking approach based on biochemical or biophysical information. J. Am. Chem. Soc. 125, 1731–1737 (2003).

    Article  CAS  Google Scholar 

  38. Woodhull, A.M. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61, 687–708 (1973).

    Article  CAS  Google Scholar 

  39. Lenaeus, M.J., Vamvouka, M., Focia, P.J. & Gross, A. Structural basis of TEA blockade in a model potassium channel. Nat. Struct. Mol. Biol. 12, 454–459 (2005).

    Article  CAS  Google Scholar 

  40. Seidel, K. et al. Protein solid-state NMR resonance assignments from (13C,13C) correlation spectroscopy. Phys. Chem. Chem. Phys. 6, 5090–5093 (2004).

    Article  CAS  Google Scholar 

  41. Baldus, M., Petkova, A.T., Herzfeld, J. & Griffin, R.G. Cross polarization in the tilted frame: assignment and spectral simplification in heteronuclear spin systems. Mol. Phys. 95, 1197–1207 (1998).

    Article  CAS  Google Scholar 

  42. Takegoshi, K., Nakamura, S. & Terao, T. 13C-1H dipolar-assisted rotational resonance in magic-angle spinning NMR. Chem. Phys. Lett. 344, 631 (2001).

    Article  CAS  Google Scholar 

  43. Fung, B.M., Khitrin, A.K. & Ermolaev, K. An improved broadband decoupling sequence for liquid crystals and solids. J. Magn. Reson. 142, 97–101 (2000).

    Article  CAS  Google Scholar 

  44. Schüttelkopf, A.W. & van Aalten, D.M.F. PRODRG: a tool for high-throughput crystallography of protein-ligand complexes. Acta Crystallogr. D Biol. Crystallogr. 60, 1355–1363 (2004).

    Article  Google Scholar 

  45. Delcour, A.H., Martinac, B., Adler, J. & Kung, C. Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels. Biophys. J. 56, 631–636 (1989).

    Article  CAS  Google Scholar 

  46. Schrempf, H. et al. A prokaryotic potassium ion channel with two predicted transmembrane segments from Streptomyces lividans. EMBO J. 14, 5170–5178 (1995).

    Article  CAS  Google Scholar 

  47. Morris, A.L., MacArthur, M.W., Hutchinson, E.G. & Thornton, J.M. Stereochemical quality of protein structure coordinates. Proteins 12, 345–364 (1992).

    Article  CAS  Google Scholar 

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Acknowledgements

Technical assistance by B. Angerstein is gratefully acknowledged. This work was funded in part by the Deutsche Forschungsgemeinschaft (DFG; Ba 1700/9-1, Be 2345/5-1, Po 137/38-1), by a PhD fellowship to C.A. from the Stiftung Stipendien-Fonds of the Verband der Chemischen Industrie, and by a PhD fellowship to R.S. from the DFG graduate school 782 'Spectroscopy and Dynamics of Molecular Coils and Aggregates'. We are indebted to R. Wagner (University of Osnabrück, Germany) for invaluable support in context of the lipid-bilayer reconstitution experiments. We are grateful to R. Riek for providing solution-state NMR resonance assignments of KcsA constructs in micelles.

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Author notes

  1. Christian Ader and Robert Schneider: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, Göttingen, 37077, Germany

    Christian Ader, Robert Schneider, Adam Lange, Karin Giller, Stefan Becker & Marc Baldus

  2. University Hospital Hamburg-Eppendorf, Center for Molecular Neurobiology, Institute for Neural Signaltransduction, Falkenried 94, Hamburg, 20251, Germany

    Sönke Hornig, Phanindra Velisetty, Iris Ohmert & Olaf Pongs

  3. Department of Chemistry, University of California, Berkeley, 94720-1460, California, USA

    Erica M Wilson & Dirk Trauner

  4. Centre National de la Recherche Scientifique (CNRS) Institut Jean Roche, Université de la Méditerranée, Boulevard Pierre Dramard, Marseille, 13916, Cedex 20, France

    Marie-France Martin-Eauclaire

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Correspondence to Dirk Trauner, Olaf Pongs or Marc Baldus.

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Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods (PDF 521 kb)

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Ader, C., Schneider, R., Hornig, S. et al. A structural link between inactivation and block of a K+ channel. Nat Struct Mol Biol 15, 605–612 (2008). https://doi.org/10.1038/nsmb.1430

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