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.
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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.
The authors declare no competing financial interests.
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
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).
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.
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.
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).
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).
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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