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.

Molecular driving forces determining potassium channel slow inactivation

Abstract

K+ channels undergo a time-dependent slow inactivation process that plays a key role in modulating cellular excitability. Here we show that in the prokaryotic proton-gated K+ channel KcsA, the number and strength of hydrogen bonds between residues in the selectivity filter and its adjacent pore helix determine the rate and extent of C-type inactivation. Upon channel activation, the interaction between residues at positions Glu71 and Asp80 promotes filter constriction parallel to the permeation pathway, which affects K+-binding sites and presumably abrogates ion conduction. Coupling between these two positions results in a quantitative correlation between their interaction strength and the stability of the inactivated state. Engineering of these interactions in the eukaryotic voltage-dependent K+ channel Kv1.2 suggests that a similar mechanistic principle applies to other K+ channels. These observations provide a plausible physical framework for understanding C-type inactivation in K+ channels.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: The Glu71-Asp80 interaction is the driving force for inactivation in KcsA.
Figure 2: Inverse relationship between the rate of inactivation and the open dwell times.
Figure 3: Histidine at position 71 inverts the voltage dependence of inactivation.
Figure 4: Structural basis of inactivation in E71S and E71H channels.
Figure 5: Relation between the open-channel probability (evaluated from Gaussian fits of histograms of all time points) and equilibrium X71-Asp80 Cα–Cα distance extracted from molecular dynamics simulations and crystal structures.
Figure 6: Correlation between the extent of inactivation and the energetics of filter distortion.
Figure 7: Mutations at position 370 recapitulate C-type inactivation in Kv1.2.
Figure 8: Structural rearrangement during C-type inactivation at the filter.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Hoshi, T., Zagotta, W.N. & Aldrich, R.W. Two types of inactivation in Shaker K+ channels: effects of alterations in the carboxy-terminal region. Neuron 7, 547–556 (1991).

    Article  CAS  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. Yellen, G., Sodickson, D., Chen, T.Y. & Jurman, M.E. An engineered cysteine in the external mouth of a K+ channel allows inactivation to be modulated by metal binding. Biophys. J. 66, 1068–1075 (1994).

    Article  CAS  Google Scholar 

  4. Liu, Y., Jurman, M.E. & Yellen, G. Dynamic rearrangement of the outer mouth of a K+ channel during gating. Neuron 16, 859–867 (1996).

    Article  CAS  Google Scholar 

  5. Kiss, L., LoTurco, J. & Korn, S.J. Contribution of the selectivity filter to inactivation in potassium channels. Biophys. J. 76, 253–263 (1999).

    Article  CAS  Google Scholar 

  6. Choi, K.L., Aldrich, R.W. & Yellen, G. Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels. Proc. Natl. Acad. Sci. USA 88, 5092–5095 (1991).

    Article  CAS  Google Scholar 

  7. Lopez-Barneo, J., Hoshi, T., Heinemann, S.H. & Aldrich, R.W. Effects of external cations and mutations in the pore region on C-type inactivation of Shaker potassium channels. Receptors Channels 1, 61–71 (1993).

    CAS  PubMed  Google Scholar 

  8. Demo, S.D. & Yellen, G. Ion effects on gating of the Ca2+-activated K+ channel correlate with occupancy of the pore. Biophys. J. 61, 639–648 (1992).

    Article  CAS  Google Scholar 

  9. Kurata, H.T. & Fedida, D. A structural interpretation of voltage-gated potassium channel inactivation. Prog. Biophys. Mol. Biol. 92, 185–208 (2006).

    Article  CAS  Google Scholar 

  10. Seebohm, G., Sanguinetti, M.C. & Pusch, M. Tight coupling of rubidium conductance and inactivation in human KCNQ1 potassium channels. J. Physiol. (Lond.) 552, 369–378 (2003).

    Article  CAS  Google Scholar 

  11. Chapman, M.L., Blanke, M.L., Krovetz, H.S. & Vandongen, A.M. Allosteric effects of external K+ ions mediated by the aspartate of the GYGD signature sequence in the Kv2.1 K+ channel. Pflugers Arch. 451, 776–792 (2006).

    Article  CAS  Google Scholar 

  12. Ficker, E., Jarolimek, W., Kiehn, J., Baumann, A. & Brown, A.M. Molecular determinants of dofetilide block of HERG K+ channels. Circ. Res. 82, 386–395 (1998).

    Article  CAS  Google Scholar 

  13. Yifrach, O. & MacKinnon, R. Energetics of pore opening in a voltage-gated K+ channel. Cell 111, 231–239 (2002).

    Article  CAS  Google Scholar 

  14. Lu, T. et al. Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter. Nat. Neurosci. 4, 239–246 (2001).

    Article  CAS  Google Scholar 

  15. Alagem, N., Yesylevskyy, S. & Reuveny, E. The pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels. Biophys. J. 85, 300–312 (2003).

    Article  CAS  Google Scholar 

  16. Gao, L., Mi, X., Paajanen, V., Wang, K. & Fan, Z. Activation-coupled inactivation in the bacterial potassium channel KcsA. Proc. Natl. Acad. Sci. USA 102, 17630–17635 (2005).

    Article  CAS  Google Scholar 

  17. 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 

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

    Article  CAS  Google Scholar 

  19. 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 

  20. Kuo, A. et al. Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300, 1922–1926 (2003).

    Article  CAS  Google Scholar 

  21. Yang, J., Yu, M., Jan, Y.N. & Jan, L.Y. Stabilization of ion selectivity filter by pore loop ion pairs in an inwardly rectifying potassium channel. Proc. Natl. Acad. Sci. USA 94, 1568–1572 (1997).

    Article  CAS  Google Scholar 

  22. Perozo, E., Cortes, D.M. & Cuello, L.G. Three-dimensional architecture and gating mechanism of a K+ channel studied by EPR spectroscopy. Nat. Struct. Biol. 5, 459–469 (1998).

    Article  CAS  Google Scholar 

  23. Perozo, E., Cortes, D.M. & Cuello, L.G. Structural rearrangements underlying K+-channel activation gating. Science 285, 73–78 (1999).

    Article  CAS  Google Scholar 

  24. Baukrowitz, T. & Yellen, G. Modulation of K+ current by frequency and external [K+]: a tale of two inactivation mechanisms. Neuron 15, 951–960 (1995).

    Article  CAS  Google Scholar 

  25. Panyi, G. & Deutsch, C. Cross talk between activation and slow inactivation gates of Shaker potassium channels. J. Gen. Physiol. 128, 547–559 (2006).

    Article  CAS  Google Scholar 

  26. Smith, P.L., Baukrowitz, T. & Yellen, G. The inward rectification mechanism of the HERG cardiac potassium channel. Nature 379, 833–836 (1996).

    Article  CAS  Google Scholar 

  27. Torrie, G.M. & Valleau, J.P. Nonphysical sampling distributions in Monte Carlo free-energy estimation: umbrella sampling. J. Comp. Phys. 23, 187–199 (1977).

    Article  Google Scholar 

  28. 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 

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

    Article  CAS  Google Scholar 

  30. Stuhmer, W. et al. Molecular basis of functional diversity of voltage-gated potassium channels in mammalian brain. EMBO J. 8, 3235–3244 (1989).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Spector, P.S., Curran, M.E., Zou, A., Keating, M.T. & Sanguinetti, M.C. Fast inactivation causes rectification of the IKr channel. J. Gen. Physiol. 107, 611–619 (1996).

    Article  CAS  Google Scholar 

  33. Sun, Z.P., Akabas, M.H., Goulding, E.H., Karlin, A. & Siegelbaum, S.A. Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating. Neuron 16, 141–149 (1996).

    Article  CAS  Google Scholar 

  34. Bruening-Wright, A., Schumacher, M.A., Adelman, J.P. & Maylie, J. Localization of the activation gate for small conductance Ca2+-activated K+ channels. J. Neurosci. 22, 6499–6506 (2002).

    Article  CAS  Google Scholar 

  35. Claydon, T.W., Makary, S.Y., Dibb, K.M. & Boyett, M.R. The selectivity filter may act as the agonist-activated gate in the G protein-activated Kir3.1/Kir3.4 K+ channel. J. Biol. Chem. 278, 50654–50663 (2003).

    Article  CAS  Google Scholar 

  36. 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 

  37. Swenson, R.P., Jr & Armstrong, C.M. K+ channels close more slowly in the presence of external K+ and Rb+. Nature 291, 427–429 (1981).

    Article  CAS  Google Scholar 

  38. Spruce, A.E., Standen, N.B. & Stanfield, P.R. Rubidium ions and the gating of delayed rectifier potassium channels of frog skeletal muscle. J. Physiol. (Lond.) 411, 597–610 (1989).

    Article  CAS  Google Scholar 

  39. Chapman, M.L., VanDongen, H.M. & VanDongen, A.M. Activation-dependent subconductance levels in the drk1 K channel suggest a subunit basis for ion permeation and gating. Biophys. J. 72, 708–719 (1997).

    Article  CAS  Google Scholar 

  40. Zheng, J. & Sigworth, F.J. Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels. J. Gen. Physiol. 112, 457–474 (1998).

    Article  CAS  Google Scholar 

  41. Proks, P., Capener, C.E., Jones, P. & Ashcroft, F.M. Mutations within the P-loop of Kir6.2 modulate the intraburst kinetics of the ATP-sensitive potassium channel. J. Gen. Physiol. 118, 341–353 (2001).

    Article  CAS  Google Scholar 

  42. 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 

  43. Claydon, T.W. et al. A direct demonstration of closed-state inactivation of K+ channels at low pH. J. Gen. Physiol. 129, 437–455 (2007).

    Article  CAS  Google Scholar 

  44. Cortes, D.M. & Perozo, E. Structural dynamics of the Streptomyces lividans K+ channel (SKC1): oligomeric stoichiometry and stability. Biochemistry 36, 10343–10352 (1997).

    Article  CAS  Google Scholar 

  45. 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 

  46. 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 

  47. Otwinowski, Z. & Minor, W. Macromolecular crystallography, part A. Methods Enzymol. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  48. Jones, T.A., Zou, J.-Y., Cowan, S.W. & Kjeldgaard, M. Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  50. Brooks, B.R. et al. CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187–217 (1983).

    Article  CAS  Google Scholar 

  51. Berneche, S. & Roux, B. Molecular dynamics of the KcsA K+ channel in a bilayer membrane. Biophys. J. 78, 2900–2917 (2000).

    Article  CAS  Google Scholar 

  52. Kumar, S., Bouzida, D., Swendsen, R.H., Kollman, P.A. & Rosenberg, J.M. The weighted histogram analysis method for free-energy calculations on biomolecules. I. The methods. J. Comput. Chem. 13, 1011–1021 (1992).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F. Bezanilla, S. Chakrapani, L. Cuello, M. Sotomayor and H. Raghuraman for critical reading and discussion of the manuscript; M. Wiener and M. Purdy for crystallographic data collection (for E71T); J. Faraldo-Gomez and A. Lau for critical comments on the PMF calculations; S. Goldstein (University of Chicago) for providing access to the two electrode voltage clamp system; the staff of the GM/CA-23-ID beamline at the Advanced Photon Source for their invaluable assistance in data collection; R. MacKinnon (Rockefeller University) for providing the KcsA antibody hybridoma cell line; and the National Center for Supercomputing Applications and Jazz computing cluster at Argonne National Laboratory for computer time. This work was supported by US National Institutes of Health grants to E.P. and B.R.

Author information

Authors and Affiliations

Authors

Contributions

J.F.C.-M. carried out the channel mutagenesis, biochemistry, EPR studies, crystallization and electrophysiology with KcsA channels, and J.F.C.-M. and A.L. carried these out on Kv1.2 channels. V.J. carried out crystal data collection, structure solving and computational analyses. V.V. participated in channel mutagenesis, biochemistry and EPR. D.M.C. made all the Fab preparations. B.R., working with V.J., participated in computational design and PMF calculations. E.P. directed experimental design and data analyses, and wrote the manuscript with J.F.C.-M., V.J. and B.R.

Corresponding author

Correspondence to Eduardo Perozo.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1–6, Supplementary Table 1 (PDF 4408 kb)

Supplementary Video 1

E71H movie. These movies were made with VMD. VMD is developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign. (http://www.ks.uiuc.edu/)7. All the molecular graphic figures in this work were made using Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) http://pymol.sourceforge.net/). (MPG 3664 kb)

Supplementary Video 2

E71A movie. These movies were made with VMD. VMD is developed with NIH support by the Theoretical and Computational Biophysics group at the Beckman Institute, University of Illinois at Urbana-Champaign. (http://www.ks.uiuc.edu/)7. All the molecular graphic figures in this work were made using Pymol (DeLano, W.L. The PyMOL Molecular Graphics System (2002) http://pymol.sourceforge.net/). (MPG 3445 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cordero-Morales, J., Jogini, V., Lewis, A. et al. Molecular driving forces determining potassium channel slow inactivation. Nat Struct Mol Biol 14, 1062–1069 (2007). https://doi.org/10.1038/nsmb1309

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb1309

This article is cited by

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