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Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel

Abstract

Na+-activated K+ channels are members of the Slo family of large conductance K+ channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels fulfil a number of biological roles and have intriguing biophysical properties, including conductance levels that are ten times those of most other K+ channels and gating sensitivity to intracellular Na+. Here we present the structure of a complete Na+-activated K+ channel, chicken Slo2.2, in the Na+-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 ångströms. The channel is composed of a large cytoplasmic gating ring, in which resides the Na+-binding site and a transmembrane domain that closely resembles voltage-gated K+ channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure reveals features that can explain the unusually high conductance of Slo channels and how contraction of the cytoplasmic gating ring closes the pore.

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Figure 1: Cryo-EM structure of chicken Slo2.2.
Figure 2: Architecture of Slo2.2.
Figure 3: Interactions between pore and S1–S4 domains.
Figure 4: Slo2.2 ion conduction pathway.
Figure 5: Slo2.2 gating ring.
Figure 6: Slo2.2 gating.

Accession codes

Primary accessions

Electron Microscopy Data Bank

Protein Data Bank

Data deposits

The 3D cryo-EM density maps of Slo2.2 with low-pass filter and amplitude modification have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-3062 (Slo2.2 whole channel), EMD-3063 (Slo2.2 gating ring) and EMD-3064 (Slo2.2 TMD). Atomic coordinates for the atomic model of full-length Slo2.2, Slo2.2 gating ring and Slo2.2 TMD have been deposited in the Protein Data Bank under accession numbers 5A6E, 5A6F and 5A6G, respectively.

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Acknowledgements

We thank Z. Yu and J. de la Cruz at the Howard Hughes Medical Institute Janelia Cryo-EM facility for assistance in data collection, S. Harrison and S. Jenni for assistance with Phenix refinement of cryo-EM density maps, and members of the MacKinnon laboratory for discussions. This work used the Extreme Science and Engineering Discovery Environment (XSEDE), which is supported by National Science Foundation grant number ACI-1053575. 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 T.W. and R.M. are investigators of the Howard Hughes Medical Institute.

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Contributions

R.K.H. performed the experiments. P.Y. provided assistance with protein expression and purification. Z.L. aided with sample preparation and data collection. Y.H. provided assistance with protein expression. T.W. aided with initial model generation and map interpretation. R.K.H and R.M. designed the experiments and analysed the results. R.K.H. and R.M. prepared the manuscript with input from all co-authors.

Corresponding author

Correspondence to Roderick MacKinnon.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Sequence alignment of Slo channels.

a, Sequence alignment of chicken Slo2.2 with human Slo2.2 and human Slo2.1. b, Predicted position of transmembrane helices in Slo2.2 S1–S4 domain on the basis of hydropothy analysis using Jpred 4 (ref. 72). c, d, Structure-based sequence alignment of chicken Slo2.2 TMD with rat Kv chimaera (c) and chicken Slo2.2 gating ring with human Slo1 gating ring (d). Helices are blue and β-strands are red.

Extended Data Figure 2 Full channel 3D reconstruction of chicken Slo2.2.

a, Representative micrograph of detergent- and lipid-solubilized Slo2.2 in vitreous ice. b, Selected 2D class averages. c, Ab initio model of Slo2.2. d, FSC curve of the full channel reconstruction with the nominal resolution estimated to be 4.5 Å on the basis of the FSC = 0.143 (dashed line) cut-off criterion.

Extended Data Figure 3 Focused refinement of the gating ring and the TMD.

a, 3D density map of the full channel reconstruction, coloured according to local resolution (in ångströms). b, c, 3D density map calculated following focused refinement using a mask to only include the gating ring (b) and the TMD (c), coloured according to local resolution (in ångströms). d, FSC of the full channel reconstruction (estimated resolution of 4.5 Å), the gating-ring-focused refinement reconstruction (4.2 Å) and the TMD-focused refinement reconstruction (5.2 Å).

Extended Data Figure 4 Validation of the Slo2.2 model.

a, Refinement statistics for the Slo2.2 full channel, TMD and gating-ring models. b, c, FSC curves for cross-validation of the refined gating ring (b) and TMD (c) models. The black curves are the refined model compared to the full data set, the red curves are the refined model compared to half map 1 (used during test refinement) and the blue curves are the refined model compared to half map 2 (not used during test refinement).

Extended Data Figure 5 K+ ions in Slo2.2.

a, Central section of the density maps of the two independently calculated half maps (coloured in green and red) with densities corresponding to K+ ions labelled. b, Superposition of the Slo2.2 selectivity filter (green) with KcsA (PDB code 1K4C) selectivity filter (yellow). Density peaks resolved in the Slo2.2 selectivity filter at 6.5 σ are shown as blue meshes. K+ ions resolved in KcsA are shown as grey spheres.

Extended Data Figure 6 Representative segments of the cryo-EM density map.

ad, Selected regions of the gating-ring density (a, b) and the TMD density (c, d) maps with the refined model.

Extended Data Figure 7 Single channel conductance of Slo2.2.

a, Single channel current–voltage relationship (mean ± s.e.m.) for Slo2.2 in planar lipid bilayers. Single channel conductance is about 200 pS. b, Representative recordings of Slo2.2 held at −80 mV, −40 mV and 0 mV in planar lipid bilayers. Chamber solution contained 135 mM NaCl and 15 mM KCl, and cup solution contained 150 mM KCl. c, Histogram of Slo2.2 currents when held at −80 mV, −40 mV and 0 mV, as labelled.

Extended Data Figure 8 Inner helix gate.

a, Ribbon diagram of the Slo2.2 pore with Met333 side chains modelled as spheres. b, Pore radius plot as a function of distance from the extracellular surface for Slo2.2 with Met333 modelled as each of the six most frequently observed rotamers, as labelled. For distances less than about 40 Å, the curves coincide.

Extended Data Figure 9 Slo2.2 gating ring is in a closed conformation.

Wire diagrams of Slo1 gating ring in the open (top left) and closed (top right) conformations. The mobile RCK1 N lobe is black and the rest of the gating ring is grey. The N-terminal residue of the gating ring, Lys343, is shown as a pink sphere. Wire diagram of the Slo2.2 gating ring (bottom) with the RCK1 N-lobe blue and the rest of the gating ring light blue. The N-terminal residue of the gating ring, Lys351, is shown as a pink sphere.

Extended Data Table 1 3D reconstructions of chicken Slo2.2 by cryo-EM

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Hite, R., Yuan, P., Li, Z. et al. Cryo-electron microscopy structure of the Slo2.2 Na+-activated K+ channel. Nature 527, 198–203 (2015). https://doi.org/10.1038/nature14958

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