Article | Published:

Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel

Nature volume 541, pages 4651 (05 January 2017) | Download Citation

Abstract

The Ca2+-activated K+ channel, Slo1, has an unusually large conductance and contains a voltage sensor and multiple chemical sensors. Dual activation by membrane voltage and Ca2+ renders Slo1 central to processes that couple electrical signalling to Ca2+-mediated events such as muscle contraction and neuronal excitability. Here we present the cryo-electron microscopy structure of a full-length Slo1 channel from Aplysia californica in the presence of Ca2+ and Mg2+ at a resolution of 3.5 Å. The channel adopts an open conformation. Its voltage-sensor domain adopts a non-domain-swapped attachment to the pore and contacts the cytoplasmic Ca2+-binding domain from a neighbouring subunit. Unique structural features of the Slo1 voltage sensor suggest that it undergoes different conformational changes than other known voltage sensors. The structure reveals the molecular details of three distinct divalent cation-binding sites identified through electrophysiological studies of mutant Slo1 channels.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

Protein Data Bank

References

  1. 1.

    , & Single channel recordings of Ca2+-activated K+ currents in rat muscle cell culture. Nature 293, 471–474 (1981)

  2. 2.

    Ca-dependent K channels with large unitary conductance in chromaffin cell membranes. Nature 291, 497–500 (1981)

  3. 3.

    , & 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)

  4. 4.

    , & Properties of single calcium-activated potassium channels in cultured rat muscle. J. Physiol. (Lond.) 331, 211–230 (1982)

  5. 5.

    et al. A BK (Slo1) channel journey from molecule to physiology. Channels (Austin) 7, 442–458 (2013)

  6. 6.

    , , , & Structure of the RCK domain from the E. coli K+ channel and demonstration of its presence in the human BK channel. Neuron 29, 593–601 (2001)

  7. 7.

    , & Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309, 897–903 (2005)

  8. 8.

    , , & Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment. Nature 450, 376–382 (2007)

  9. 9.

    , , & Structure of the gating ring from the human large-conductance Ca2+-gated K+ channel. Nature 466, 393–397 (2010)

  10. 10.

    , , , & Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution. Science 329, 182–186 (2010)

  11. 11.

    , , & Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel. Nature 481, 94–97 (2011)

  12. 12.

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

  13. 13.

    , & Multiple regulatory sites in large-conductance calcium-activated potassium channels. Nature 418, 880–884 (2002)

  14. 14.

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

  15. 15.

    , , & Elimination of the BKCa channel’s high-affinity Ca2+ sensitivity. J. Gen. Physiol. 120, 173–189 (2002)

  16. 16.

    , , & Mapping the BKCa channel’s “Ca2+ bowl”: side-chains essential for Ca2+ sensing. J. Gen. Physiol. 123, 475–489 (2004)

  17. 17.

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

  18. 18.

    , & Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane. J. Gen. Physiol. 92, 67–86 (1988)

  19. 19.

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

  20. 20.

    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)

  21. 21.

    et al. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol. 45, 1227–1234 (1994)

  22. 22.

    et al. The appearance of a protein kinase A-regulated splice isoform of slo is associated with the maturation of neurons that control reproductive behavior. J. Biol. Chem. 279, 52324–52330 (2004)

  23. 23.

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

  24. 24.

    et al. Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo). Proc. Natl Acad. Sci. USA 94, 5427–5431 (1997)

  25. 25.

    & Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca2+. J. Gen. Physiol. 114, 305–336 (1999)

  26. 26.

    , & Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca2+. J. Gen. Physiol. 114, 277–304 (1999)

  27. 27.

    The variance of sodium current fluctuations at the node of Ranvier. J. Physiol. (Lond.) 307, 97–129 (1980)

  28. 28.

    , & Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309, 903–908 (2005)

  29. 29.

    , , , & A gating charge transfer center in voltage sensors. Science 328, 67–73 (2010)

  30. 30.

    et al. Mechanism of voltage gating in potassium channels. Science 336, 229–233 (2012)

  31. 31.

    , & Calibrated measurement of gating-charge arginine displacement in the KvAP voltage-dependent K+ channel. Cell 123, 463–475 (2005)

  32. 32.

    et al. Location of modulatory beta subunits in BK potassium channels. J. Gen. Physiol. 135, 449–459 (2010)

  33. 33.

    & The occupancy of ions in the K+ selectivity filter: charge balance and coupling of ion binding to a protein conformational change underlie high conduction rates. J. Mol. Biol. 333, 965–975 (2003)

  34. 34.

    & Structural basis of inward rectification: cytoplasmic pore of the G protein-gated inward rectifier GIRK1 at 1.8 Å resolution. Cell 111, 957–965 (2002)

  35. 35.

    et al. Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel. Nature 527, 198–203 (2015)

  36. 36.

    , & A ring of eight conserved negatively charged amino acids doubles the conductance of BK channels and prevents inward rectification. Proc. Natl Acad. Sci. USA 100, 9017–9022 (2003)

  37. 37.

    , & Electrostatic tuning of ion conductance in potassium channels. Biochemistry 42, 9263–9268 (2003)

  38. 38.

    , , , & Properties of Slo1 K+ channels with and without the gating ring. Proc. Natl Acad. Sci. USA 110, 16657–16662 (2013)

  39. 39.

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

  40. 40.

    & Charge movement associated with the opening and closing of the activation gates of the Na channels. J. Gen. Physiol. 63, 533–552 (1974)

  41. 41.

    , , & Voltage-sensing residues in the S2 and S4 segments of the Shaker K+ channel. Neuron 16, 1159–1167 (1996)

  42. 42.

    & Contribution of the S4 segment to gating charge in the Shaker K+ channel. Neuron 16, 1169–1177 (1996)

  43. 43.

    , & Structural basis for gating the high-conductance Ca2+-activated K+ channel. Nature (2016)

  44. 44.

    & Intracellular Mg2+ enhances the function of BK-type Ca2+-activated K+ channels. J. Gen. Physiol . 118, 589–606 (2001)

  45. 45.

    , , & Calcium ion coordination: a comparison with that of beryllium, magnesium, and zinc. J. Am. Chem. Soc. 1, 5752–5763 (1996)

  46. 46.

    , & Coordination of water to magnesium cations. Inorg. Chem. 33, 419–427 (1994)

  47. 47.

    , & Intra- and intersubunit cooperativity in activation of BK channels by Ca2+. J. Gen. Physiol. 128, 389–404 (2006)

  48. 48.

    & Measurements of the BKCa channel’s high-affinity Ca2+ binding constants: effects of membrane voltage. J. Gen. Physiol. 132, 491–505 (2008)

  49. 49.

    et al. Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel. Nature 486, 130–134 (2012)

  50. 50.

    , , , & Crystal structure of a voltage-gated sodium channel in two potentially inactivated states. Nature 486, 135–139 (2012)

  51. 51.

    et al. Structure of the voltage-gated two-pore channel TPC1 from Arabidopsis thaliana. Nature 531, 196–201 (2016)

  52. 52.

    HOLLOW: generating accurate representations of channel and interior surfaces in molecular structures. BMC Struct. Biol. 8, (2008)

  53. 53.

    , , & A carboxy-terminal affinity tag for the purification and mass spectrometric characterization of integral membrane proteins. J. Proteome Res. 8, 2388–2396 (2009)

  54. 54.

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

  55. 55.

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

  56. 56.

    , & Quantitative analysis of mammalian GIRK2 channel regulation by G proteins, the signaling lipid PIP2 and Na+ in a reconstituted system. eLife 3, e03671 (2014)

  57. 57.

    Gating currents and charge movements in excitable membranes. Rev. Physiol. Biochem. Pharmacol . 82, 96–190 (1978)

  58. 58.

    & Survival of K+ permeability and gating currents in squid axons perfused with K+-free media. J. Gen. Physiol. 75, 61–78 (1980)

  59. 59.

    & Voltage-dependent gating of ionic channels. Annu. Rev. Biophys. Biomol. Struct. 23, 819–846 (1994)

  60. 60.

    , , & Transfer of twelve charges is needed to open skeletal muscle Na+ channels. J. Gen. Physiol. 106, 1053–1068 (1995)

  61. 61.

    et al. Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels. J. Gen. Physiol. 108, 143–155 (1996)

  62. 62.

    & Total charge movement per channel. The relation between gating charge displacement and the voltage sensitivity of activation. J. Gen. Physiol . 109, 27–39 (1997)

  63. 63.

    & Channel noise in nerve membranes and lipid bilayers. Q. Rev. Biophys. 8, 451–506 (1975)

  64. 64.

    Sodium channels in nerve apparently have two conductance states. Nature 270, 265–267 (1977)

  65. 65.

    Empirical considerations regarding the use of ensemble-variance analysis of macroscopic currents. J. Neurosci. Methods 158, 121–132 (2006)

  66. 66.

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

  67. 67.

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

  68. 68.

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

  69. 69.

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

  70. 70.

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

  71. 71.

    , , & High resolution single particle refinement in EMAN2.1. Methods 100, 25–34 (2016)

  72. 72.

    , , & Likelihood-based classification of cryo-EM images using FREALIGN. J. Struct. Biol. 183, 377–388 (2013)

  73. 73.

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

  74. 74.

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

  75. 75.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  76. 76.

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

  77. 77.

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

  78. 78.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)

  79. 79.

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

Download references

Acknowledgements

We thank M. Ebrahim for assistance in data collection; R. W. Aldrich for comments on the manuscript; and members of the MacKinnon lab 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

Affiliations

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

    • Xiao Tao
    • , Richard K. Hite
    •  & Roderick MacKinnon

Authors

  1. Search for Xiao Tao in:

  2. Search for Richard K. Hite in:

  3. Search for Roderick MacKinnon in:

Contributions

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

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Roderick MacKinnon.

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

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature20608

Further reading

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.