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:

Structural basis for gating the high-conductance Ca2+-activated K+ channel

Nature volume 541, pages 52–57 (2017)Cite this article

Subjects

Abstract

The precise control of an ion channel gate by environmental stimuli is crucial for the fulfilment of its biological role. The gate in Slo1 K+ channels is regulated by two separate stimuli, intracellular Ca2+ concentration and membrane voltage. Slo1 is thus central to understanding the relationship between intracellular Ca2+ and membrane excitability. Here we present the Slo1 structure from Aplysia californica in the absence of Ca2+ and compare it with the Ca2+-bound channel. We show that Ca2+ binding at two unique binding sites per subunit stabilizes an expanded conformation of the Ca2+ sensor gating ring. These conformational changes are propagated from the gating ring to the pore through covalent linkers and through protein interfaces formed between the gating ring and the voltage sensors. The gating ring and the voltage sensors are directly connected through these interfaces, which allow membrane voltage to regulate gating of the pore by influencing the Ca2+ sensors.

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: Comparison of Ca2+-bound and EDTA Slo1 structures.
Figure 2: Comparison of the Ca2+-binding sites.
Figure 3: Interdomain interfaces.
Figure 4: Voltage-sensor–RCK1 N-lobe interface.
Figure 5: Stereo view of the voltage-sensor domain.
Figure 6: Model of Slo1 gating by Ca2+ and voltage.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Hille, B. Ion Channels of Excitable Membranes 3rd edn (Sinauer, 2001)

  2. Pallotta, B. S., Magleby, K. L. & Barrett, J. N. Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature 293, 471–474 (1981)

    Article  CAS  ADS  Google Scholar 

  3. Sweet, T. B. & Cox, D. H. Measurements of the BKCa channel’s high-affinity Ca2+ binding constants: effects of membrane voltage. J. Gen. Physiol. 132, 491–505 (2008)

    Article  CAS  Google Scholar 

  4. Tao, X., Hite, R. K. & MacKinnon, R. Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature http://dx.doi.org/10.1038/nature20608 (2016)

  5. Horrigan, F. T. & Aldrich, R. W. Coupling between voltage sensor activation, Ca2+ binding and channel opening in large conductance (BK) potassium channels. J. Gen. Physiol. 120, 267–305 (2002)

    Article  CAS  Google Scholar 

  6. McManus, O. B. & Magleby, K. L. Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle. J. Physiol. (Lond.) 402, 79–120 (1988)

    Article  CAS  Google Scholar 

  7. Qian, X., Niu, X. & Magleby, K. L. Intra- and intersubunit cooperativity in activation of BK channels by Ca2+ . J. Gen. Physiol. 128, 389–404 (2006)

    Article  CAS  Google Scholar 

  8. Savalli, N., Pantazis, A., Yusifov, T., Sigg, D. & Olcese, R. The contribution of RCK domains to human BK channel allosteric activation. J. Biol. Chem. 287, 21741–21750 (2012)

    Article  CAS  Google Scholar 

  9. Carrasquel-Ursulaez, W. et al. Hydrophobic interaction between contiguous residues in the S6 transmembrane segment acts as a stimuli integration node in the BK channel. J. Gen. Physiol. 145, 61–74 (2015)

    Article  CAS  Google Scholar 

  10. Wu, Y., Yang, Y., Ye, S. & Jiang, Y. Structure of the gating ring from the human large-conductance Ca2+-gated K+ channel. Nature 466, 393–397 (2010)

    Article  CAS  ADS  Google Scholar 

  11. Yuan, P., Leonetti, M. D., Hsiung, Y. & MacKinnon, R. Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature 481, 94–97 (2011)

    Article  ADS  Google Scholar 

  12. Latorre, R., Vergara, C. & Hidalgo, C. Reconstitution in planar lipid bilayers of a Ca2+-dependent K+ channel from transverse tubule membranes isolated from rabbit skeletal muscle. Proc. Natl Acad. Sci. USA 79, 805–809 (1982)

    Article  CAS  ADS  Google Scholar 

  13. Moczydlowski, E. & Latorre, R. Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. J. Gen. Physiol. 82, 511–542 (1983)

    Article  CAS  Google Scholar 

  14. Niu, X., Qian, X. & Magleby, K. L. Linker-gating ring complex as passive spring and Ca2+-dependent machine for a voltage- and Ca2+-activated potassium channel. Neuron 42, 745–756 (2004)

    Article  CAS  Google Scholar 

  15. Rothberg, B. S. & Magleby, K. L. Gating kinetics of single large-conductance Ca2+-activated K+ channels in high Ca2+ suggest a two-tiered allosteric gating mechanism. J. Gen. Physiol. 114, 93–124 (1999)

    Article  CAS  Google Scholar 

  16. Golowasch, J., Kirkwood, A. & Miller, C. Allosteric effects of Mg2+ on the gating of Ca2+-activated K+ channels from mammalian skeletal muscle. J. Exp. Biol. 124, 5–13 (1986)

    CAS  PubMed  Google Scholar 

  17. Niu, X. & Magleby, K. L. Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca2+. Proc. Natl Acad. Sci. USA 99, 11441–11446 (2002)

    Article  CAS  ADS  Google Scholar 

  18. Rothberg, B. S. & Magleby, K. L. Kinetic structure of large-conductance Ca2+-activated K+ channels suggests that the gating includes transitions through intermediate or secondary states. A mechanism for flickers. J. Gen. Physiol. 111, 751–780 (1998)

    Article  CAS  Google Scholar 

  19. Yang, H. et al. Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1 domains. Nat. Struct. Mol. Biol. 15, 1152–1159 (2008)

    Article  CAS  Google Scholar 

  20. Yang, H. et al. Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK channels. Proc. Natl Acad. Sci. USA 104, 18270–18275 (2007)

    Article  CAS  ADS  Google Scholar 

  21. Horrigan, F. T. & Ma, Z. Mg2+ enhances voltage sensor/gate coupling in BK channels. J. Gen. Physiol. 131, 13–32 (2008)

    Article  CAS  Google Scholar 

  22. Yang, J. et al. Interaction between residues in the Mg2+-binding site regulates BK channel activation. J. Gen. Physiol. 141, 217–228 (2013)

    Article  CAS  ADS  Google Scholar 

  23. Shi, J. et al. Mechanism of magnesium activation of calcium-activated potassium channels. Nature 418, 876–880 (2002)

    Article  CAS  ADS  Google Scholar 

  24. Budelli, G., Geng, Y., Butler, A., Magleby, K. L. & Salkoff, L. Properties of Slo1 K+ channels with and without the gating ring. Proc. Natl Acad. Sci. USA 110, 16657–16662 (2013)

    Article  CAS  ADS  Google Scholar 

  25. Horrigan, F. T. Perspectives on: conformational coupling in ion channels: conformational coupling in BK potassium channels. J. Gen. Physiol. 140, 625–634 (2012)

    Article  CAS  Google Scholar 

  26. Schreiber, M. & Salkoff, L. A novel calcium-sensing domain in the BK channel. Biophys. J. 73, 1355–1363 (1997)

    Article  CAS  Google Scholar 

  27. Xia, X. M., Zeng, X. & Lingle, C. J. Multiple regulatory sites in large-conductance calcium-activated potassium channels. Nature 418, 880–884 (2002)

    Article  CAS  ADS  Google Scholar 

  28. Zhang, G. et al. Ion sensing in the RCK1 domain of BK channels. Proc. Natl Acad. Sci. USA 107, 18700–18705 (2010)

    Article  CAS  ADS  Google Scholar 

  29. Magleby, K. L. & Song, L. Dependency plots suggest the kinetic structure of ion channels. Proc. R. Soc. Lond. B 249, 133–142 (1992)

    Article  CAS  ADS  Google Scholar 

  30. Rothberg, B. S., Bello, R. A. & Magleby, K. L. Two-dimensional components and hidden dependencies provide insight into ion channel gating mechanisms. Biophys. J. 72, 2524–2544 (1997)

    Article  CAS  Google Scholar 

  31. Bao, L., Rapin, A. M., Holmstrand, E. C. & Cox, D. H. Elimination of the BKCa channel’s high-affinity Ca2+ sensitivity. J. Gen. Physiol. 120, 173–189 (2002)

    Article  CAS  Google Scholar 

  32. Li, W. & Aldrich, R. W. Unique inner pore properties of BK channels revealed by quaternary ammonium block. J. Gen. Physiol. 124, 43–57 (2004)

    Article  CAS  Google Scholar 

  33. Wilkens, C. M. & Aldrich, R. W. State-independent block of BK channels by an intracellular quaternary ammonium. J. Gen. Physiol. 128, 347–364 (2006)

    Article  CAS  Google Scholar 

  34. Geng, Y., Niu, X. & Magleby, K. L. Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume. J. Gen. Physiol. 137, 533–548 (2011)

    Article  CAS  Google Scholar 

  35. Zhou, Y., Xia, X. M. & Lingle, C. J. Cysteine scanning and modification reveal major differences between BK channels and Kv channels in the inner pore region. Proc. Natl Acad. Sci. USA 108, 12161–12166 (2011)

    Article  CAS  ADS  Google Scholar 

  36. Wong, J. P., Reboul, E., Molday, R. S. & Kast, J. A carboxy-terminal affinity tag for the purification and mass spectrometric characterization of integral membrane proteins. J. Proteome Res. 8, 2388–2396 (2009)

    Article  CAS  Google Scholar 

  37. Rothbauer, U. et al. A versatile nanotrap for biochemical and functional studies with fluorescent fusion proteins. Mol. Cell. Proteomics 7, 282–289 (2008)

    Article  CAS  Google Scholar 

  38. Fridy, P. C. et al. A robust pipeline for rapid production of versatile nanobody repertoires. Nat. Methods 11, 1253–1260 (2014)

    Article  CAS  Google Scholar 

  39. Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005)

    Article  Google Scholar 

  40. Grant, T. & Grigorieff, N. Measuring the optimal exposure for single particle cryo-EM using a 2.6 Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015)

    Article  Google Scholar 

  41. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

    Article  CAS  Google Scholar 

  42. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

    Article  Google Scholar 

  43. Bell, J. M., Chen, M., Baldwin, P. R. & Ludtke, S. J. High resolution single particle refinement in EMAN2.1. Methods 100, 25–34 (2016)

    Article  CAS  Google Scholar 

  44. Lyumkis, D., Brilot, A. F., Theobald, D. L. & Grigorieff, N. Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377–388 (2013)

    Article  CAS  Google Scholar 

  45. Rosenthal, P. B. & Henderson, R. Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. J. Mol. Biol. 333, 721–745 (2003)

    Article  CAS  Google Scholar 

  46. Rubinstein, J. L. & Brubaker, M. A. Alignment of cryo-EM movies of individual particles by optimization of image translations. J. Struct. Biol. 192, 188–195 (2015)

    Article  Google Scholar 

  47. Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)

    Article  CAS  Google Scholar 

  48. Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

    Article  CAS  Google Scholar 

  49. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

    Article  CAS  Google Scholar 

  50. Adams, P. D. et al. The Phenix software for automated determination of macromolecular structures. Methods 55, 94–106 (2011)

    Article  CAS  Google Scholar 

  51. Brown, A. et al. Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions. Acta Crystallogr. D 71, 136–153 (2015)

    Article  CAS  Google Scholar 

  52. Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013)

    Article  Google Scholar 

Download references

Acknowledgements

We thank Z. Yu and R. Huang at the Howard Hughes Medical Institute Janelia Cryo-EM facility for assistance in data collection; R. W. Aldrich for comments on the manuscript and members of the MacKinnon laboratory for assistance. This work was supported in part by GM43949. R.K.H. is a Howard Hughes Medical Institute postdoctoral fellow of the Helen Hay Whitney Foundation and R.M. is an investigator of the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Rockefeller University and Howard Hughes Medical Institute, 1230 York Avenue, New York, 10065, New York, USA

    Richard K. Hite, Xiao Tao & Roderick MacKinnon

Authors
  1. Richard K. Hite
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Xiao Tao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Roderick MacKinnon
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.K.H. and X.T. performed the experiments. R.K.H., X.T. and R.M. designed the experiments, analysed the results and prepared the manuscript.

Corresponding author

Correspondence to Roderick MacKinnon.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks F. Horrigan, K. Magleby and J. Rubinstein for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Cryo-EM reconstructions of Aplysia Slo1 in four EDTA classes.

a, Representative image and 2D class averages of vitrified Aplysia Slo1. Scale bar, 500 Å. b, Angular distribution plot for EDTA class 4 following per-particle polishing. c, Fourier shell correlation (FSC) curves for Aplysia Slo1 in four EDTA states with overall resolutions estimated to be 3.88 Å (EDTA 1, blue), 3.84 Å (EDTA 2, green), 4.02 Å (EDTA 3, pink), 4.02 Å (EDTA 4, red) and 3.76 Å (EDTA 4 following per-particle polishing) on the basis of the FSC = 0.143 (dashed line) cut-off criterion. d–h, Section of the cryo-EM density maps of EDTA 1 (d), EDTA 2 (e), EDTA 3 (f), EDTA 4 (g) and EDTA 4 following per-particle polishing (h) coloured by local resolution (in ångstroms).

Extended Data Figure 2 Cryo-EM structure of EDTA Aplysia Slo1.

a, Cryo-EM density maps of two conformations of EDTA Slo1 aligned by their gating rings. The density slices correspond to the regions between the dashed lines in the TMD and the gating ring. b, Wire diagrams of EDTA Slo1 in four different conformations. c, Superposition of two EDTA Slo1 conformations aligned by their pore helices and selectivity filters. d, Superposition of the inner (S6) helices of the four EDTA Slo1 states aligned by their pore helices and selectivity filters. e, Plot of pore diameter along the length of the pore in the TMD for the four EDTA and the open Slo1 states.

Extended Data Figure 3 Cryo-EM reconstruction of Aplysia Slo1 gating ring in the EDTA state.

a, Cryo-EM density map of Slo1 gating ring in the EDTA state from the full channel reconstruction and following focused refinement. The mask used for focused refinement is shown as a grey transparent surface. b, Fourier shell correlation curves for focused refinement of the Slo1 gating ring with overall resolution estimated to be 3.46 Å on the basis of the FSC = 0.143 (dashed line) cut-off criterion.

Extended Data Figure 4 Comparison of cryo-EM reconstructions of Aplysia Slo1 in the Ca2+-bound and EDTA states.

Sections of cryo-EM density of Aplysia Slo1 in the Ca2+-bound (blue) and the EDTA (red) states aligned by their TMDs.

Extended Data Figure 5 Comparison of the Aplysia Slo1 EDTA state with closed human Slo1 gating ring.

Superposition the Aplysia Slo1 EDTA state (red) with closed human Slo1 gating ring (PDB code 3NAF, yellow) aligned by their RCK2 domains (grey). The spheres represent the locations of the Cα atom of Lys320 of each subunit.

Extended Data Figure 6 Validation of the refined models.

a, Refinement statistics for EDTA Slo1 models. b–g, Fourier shell correlation curves of refined model versus unmasked map for cross-validation of EDTA 1 (b), EDTA 2 (c), EDTA 3 (d), EDTA 4 (e), EDTA 4 following per-particle polishing (f) and the focused refined gating ring (g). The black curves are the refined model compared to the full dataset, the red curves are the refined model compared to half-map 1 (used during refinement) and the blue curves are the refined model compared to half-map 2 (not used during refinement).

Extended Data Figure 7 Representative segments of cryo-EM density.

a–c, Selected density fragments from the TMD (a) and gating ring (b) of EDTA 4 following per-particle polishing and the focused refinement gating ring map (c).

Supplementary information

Conformational changes to aplyisa Slo1 during Ca2+ and Mg2+ binding

This video shows the binding of Ca2+ and Mg2+ to aplysia Slo1 and the opening of the channel. (MOV 23918 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hite, R., Tao, X. & MacKinnon, R. Structural basis for gating the high-conductance Ca2+-activated K+ channel. Nature 541, 52–57 (2017). https://doi.org/10.1038/nature20775

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature20775

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