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

Nature volume 541, pages 46–51 (2017)Cite this article

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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.

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Figure 1: Dual activation of Aplysia Slo1 by voltage and intracellular Ca2+ in planar lipid bilayers.
Figure 2: Structure of open Slo1.
Figure 3: The ion conduction pathway in Aplysia Slo1.
Figure 4: Unique features of the Slo1 VSD.
Figure 5: Three divalent cation-binding sites in Aplysia Slo1.
Figure 6: Comparison of the ion conduction pore in three different K+ channels.

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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.

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Authors and Affiliations

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

    Xiao Tao, Richard K. Hite & Roderick MacKinnon

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  1. Xiao Tao
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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.

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Correspondence to Roderick MacKinnon.

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The authors declare no competing financial interests.

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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 Sequence alignment of Aplysia Slo1, human Slo1 and zebrafish (zf) Slo1.

Secondary structure elements are indicated above the sequences and disordered regions in the structure are indicated with dashed lines. Residues discussed in the text are highlighted in marine blue (Ca2+ bowl site), orange (Ca2+ RCK1 site), magenta (Mg2+ site) and cyan (selectivity filter). The three positively charged residues (R2, R3 and R4) and corresponding residues of the gating charge transfer centre are also coloured. Numbers above and below the sequences refer to the Aplysia and human Slo1, respectively.

Extended Data Figure 2 Cryo-EM density maps at the three divalent cation-binding sites.

a, Stereo view of density (grey wire mesh contoured at 6σ) near the Ca2+ bowl site and the Ca2+ RCK1 site demonstrating the structural connectivity between the two Ca2+ binding sites. The channel is shown as lines with the RCK1 domain coloured blue and RCK2 domain coloured red. The Ca2+ ions at the two sites are shown as marine blue and orange spheres, respectively. Side chains of Arg503, Glu912 and Tyr914 are shown as sticks and coloured according to atom type. b, Stereo view of density at the Mg2+ site (grey wire mesh contoured at 3σ). The channel is shown as lines with the RCK1 domain coloured blue and neighbouring VSD coloured orange. Side chains are shown as sticks and coloured according to atom type. The divalent cation is shown as a magenta sphere and water molecule as a cyan sphere.

Extended Data Figure 3 Cryo-EM reconstruction of Aplysia Slo1.

a, Representative image and 2D class averages of vitrified Aplysia Slo1. Scale bar, 500 Å. b, Angular distribution plot for Aplysia Slo1 reconstruction. c, Fourier shell correlation (FSC) curve for Aplysia Slo1. Overall resolution is estimated to be 3.47 Å on the basis of the FSC = 0.143 (dashed line) cut-off criterion. d, Cryo-EM density map coloured by local resolution using ResMap (in ångstroms)78.

Extended Data Figure 4 Representative segments of cryo-EM density.

Cryo-EM density maps are of high quality throughout the channel: density for regions of S0, S0′, S1, S2, S3, S4, S5, S6, S6–RCK1 linker, a bound partial lipid molecule, as well as representative regions of RCK1 and RCK2 domains are shown as grey wire mesh. The channel is shown as sticks and coloured according to atom type: yellow, carbon; red, oxygen; blue, nitrogen; orange, sulfur; and magenta, phosphorous.

Extended Data Figure 5 Validation of the refined model.

a, Refinement statistics for Slo1 model. b, Fourier shell correlation curves of refined model versus unmasked map for cross-validation. The black curve is the refined model compared to the full dataset, the red curve is the refined model compared to half-map 1 (used during refinement) and the blue curve is the refined model compared to half-map 2 (not used during refinement).

Extended Data Figure 6 Co-purified lipids in the structure.

a, b, Side and top views of the Slo1 transmembrane region showing the ordered lipid molecules (CPK representation, coloured according to atom type: yellow, carbon; red, oxygen; magenta, phosphorous). The channel is shown as blue Cα traces and K+ ions are shown as green spheres. c, Stereo view of detailed interactions of a lipid molecule with visible head group as well as two other partial lipids in the vicinity. Lipid molecules are shown as sticks and coloured as in a. The channel is shown as blue Cα traces and side chains involved in lipid interactions are shown as green sticks.

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Tao, X., Hite, R. & MacKinnon, R. Cryo-EM structure of the open high-conductance Ca2+-activated K+ channel. Nature 541, 46–51 (2017). https://doi.org/10.1038/nature20608

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