Open Kv1.2 overall structure.

(A) Side and top view of the Kv1.2s cryo-EM density map (upper panel) and model (lower panel). Lipid densities are colored gray in the map. (B) Side view of VSD structure with map density of Kv1.2s. (C) The relative positions of the interacting residues R2 and E138 (upper), R3 and E226 (middle), K5 and F233 (lower) are shown. (D) Superposition showing the very close match of Kv1.2 (yellow) and Shaker (gray) VSD structures. (E) Superposition of Kv1.2s (yellow) and Kv1.2-2.1 (pink) VSD structures. Positively charged, negatively charged and aromatic residues are shown as blue, red and green, respectively. VSD, voltage-sensing domain; PD, Pore domain.

Kv1.2 pore domain and selectivity filter structures in inactivated and open conformations. (A) Sequence alignment of potassium channels in the S5-S6 linker region, with the relative numbering of Miller (1990) indicated at top. (B) Side view of opposing subunits in the open Kv1.2s pore domain. Relative positions of Y28’, W17’ and D30’ are shown in the right- hand panels. Shown with a dashed blue line is the key hydrogen bond between D30’ and W17’ that is eliminated in the W17’F (W366F) mutant. (C) Side view of opposing Kv1.2s P-loops. Labels on the left show the Kv1.2 residue numbering, and on the right the Miller numbering. (D) Side view of the inactivated Kv1.2s W17’F pore domain. The new locations of Y28’, F17’ and D30’ are shown in the right panels. (E) Side view of the Kv1.2s P-loop in the inactivated conformation. In the large upper-pore cavity the G27’ and G29’ carbonyls are 5.1 and 11 Å apart. Potassium ions are shown as purple balls. (F) Superimposed top views of the Kv1.2s outer pore in inactivated (colored) and open (grey) states. In the inactivated channel the displaced Y28’ ring of one subunit is in the position occupied by D30’ - in the neighboring subunit - of the open channel. (G) The corresponding side view. The large rotation of the Y28’ side chain and flipping of D30’ are indicated by curved arrows. (H) Side view of the P-loop in inactivated (colored) and open (grey) conformations. (I,J) Details of Y28’ G27’ carbonyl and side chain reorientation from open (grey) to inactivated (colored) states are shown in side view (upper) and top view. (K) Surface renderings of open and inactivated selectivity filter region. Hydrophilic and hydrophobic surfaces are shown in teal and orange, respectively.

Kv1.2s DTx-bound structure.

(A) DTx crystal structure (pdb:1DTX) and electrostatic surface view. Basic residue sidechains are illustrated, and disulfide bridges are shown in yellow. (B) Representative 2D classes from the DTx-bound Kv1.2s. (C) Side and top view of Kv1.2s-DTx cryo-EM density map, obtained with no symmetry imposed. (D) Side and top view of the fitted model. (E) Top-down view of the Kv1.2s-DTx structure with DTx removed, shown as electrostatic surface view (upper panel); the corresponding bottom-up view through the DTx-channel interface. DTx is shown in electrostatic surface view, and extensions of Kv1.2s helices in blue. The asterisks indicate the charged groups. (F) Three salt-bridge interactions between DTX and Kv1.2s are shown as top view (upper left panel) and side views. (G) Side view of the selectivity filter of Kv1.2s-DTx. Gray dashed lines show approximate distances between carbonyl oxygens. Potassium ions are shown as blue balls.

Summary of Kv1.2 conductive and non-conductive pores. Selectivity filter structures of (A) Kv1.2s, (B) Kv1.2s Na+-bound, (C) Kv1.2s W366F, and (D) Kv1.2s-DTX. Potassium ions and sodium ions are shown as blue and orange balls, respectively. Circled numbers label the P-loop ion- coordinating oxygens. (E) Plots of density along the symmetry axis of the selectivity filter region. Values of the z-coordinate are relative to the position of the T25’ (T374) carbonyl oxygen. The nominal positions of the coordinating oxygens are marked and numbered as in parts A-D. Dashed baselines indicate the external solvent density. The scaling of the density traces is arbitrary. The DTx map was computed with no symmetry imposed; for the others, C4 symmetry was imposed in reconstruction.

Cryo-EM analysis of Kv1.2 W366F in Na+. (A) Representative 2D classes. (B) Second round of classification of first class in (A), outlined in red. Wobbling of the transmembrane domain is illustrated by the white dashed lines. (C) Density of intracellular domain, obtained by focused refinement. (D) Top view of TMD reconstruction, with resolution 7.0Å. (E) Overall TMD map density fits with the Kv1.2 W366F model, shown as ribbons. (F) There is low density in the selectivity filter (dashed rectangle).

Structural comparison of Kv1.2 W366F with various channel pores.

A-D, Selectivity filter structures and P-loop sequences of potassium selective non-conducting channels: (A) Kv1.2s W366F, (B) Kv1.3 alternate conformation, (C) Kv1.3 H451N, (D) Kv1.2-2.1 V406W. E-F, Pore regions of less-selective channels: the non- selective, conducting NaK (E) and the weakly-selective, conducting HCN (F). Potassium ions are shown as blue balls. Circled numbers enumerate the pore-forming carbonyl oxygens; carbonyl 2 faces the adjacent subunit in the clockwise direction in all but the HCN channel in panel F.

Image processing and reconstruction of Kv1.2s.

(A) Representative micrograph. (B) Representative 2D classes. (C) Cryo-EM data processing workflow. (D) Gold standard FSC resolution estimation. (F) Local resolution estimation.

currents from native and W366F Kv1.2 channels.

Xenopus oocytes were injected with mRNA for the alpha subunit constructs used in this study. A, native Kv1.2 currents elicited from pulses to -60 to + 60mV in 10 mV steps, from a holding potential of -80mV. B, Same voltage protocol applied to channels with W366F alpha subunits, recorded with 96 mM K+ bath solution. C, Comparison of currents elicited from an oocyte at +40 mV with 96mM K+ or Na+ bath solutions. The potassium-free Na+ solution yielded faster inactivation, as expected for C-type inactivation. The apparently sustained current in 95 mM Na+ solution is an artifact of P/4 leak subtraction at -120mV holding potential.

, Processing of Kv1.2 W366F images. (A) Representative micrograph. (B) Representative 2D classes. (C) Cryo-EM data processing workflow. (D) Gold standard FSC resolution estimation. (F) Local resolution estimation.

Voltage-sensing-domain conformational differences between open and C-type inactivated states. Side view of VSD structures and maps of Kv1.2s in (A) inactivated state. (B) Kv1.2s VSD R3/E183 (upper), R4/E226 (middle), and K5/F233 (lower) interactions in the inactivated state. VSD structure in open state. (D) VSD relative position of R2/E138 (upper) and R3/E226 (middle), K5/F233 (lower) interactions in the open state. Positively charged and negatively charged residues are shown in blue and red, while aromatic residues are shown in green. Side view (E), top view (F, upper) and bottom view (F, lower) VSD conformational difference between open (yellow) and inactivated (purple) states. Superposition of (G) Shaker open (PDB: 7SIP), Shaker W434F inactivated (PDB: 7SJ1) and (F) Kv1.2-2.1 open (PDB: 7SIZ), Kv1.2-2.1 3m inactivated (PDB: 7SIT) VSD structures. (I-L) Superposition of VSD structures. (I) Kv1.2s (yellow) and Kv1.2-2.1 (light blue, PDB: 2R9R); (J) Kv1.2s W366F (purple) and Kv1.2-2.1 V406W (carnation, PDB: 5WIE); (K) Kv1.2 (yellow) and Kv1.3 (green, PDB: 7EJ1); (L) Kv1.2 W366F (purple) and Kv1.3 H451N (clover, PDB: 7EJ2).

Stereo views of selectivity filter and voltage-sensing-domain. Stereo view of (A) Kv1.2s SF, (B) Kv1.2s-W366F SF, (C) Kv1.2s VSD, (D) Kv1.2s-W366F VSD.

Cryo-EM imaging and reconstruction of Kv1.2s-DTX.

(A) Representative micrograph. (B) Representative 2D classes, showing the DTx “cap” on the particles. (C) Cryo-EM data processing workflow. See Methods for details of the symmetry expansion and C1 reconstruction. (D) Gold standard FSC resolution estimation for the overall map (top) and the DTX-plus-selectivity filter masked region. (E) Local resolution estimation.

Comparison of the Kv1.2-2.1 CTx bound selectivity filter with the Kv1.2s DTx-bound structure. (A) Side view of the selectivity filter of Kv1.2-2.1 CTx bound conformation (Banerjee et al. 2013). Orange dashed lines show the distances between carbonyl oxygens, for comparison with Fig. 3G. Potassium ions are shown as blue balls. (B) Superposition of Kv1.2 DTx (red) and Kv1.2-2.1 CTx (blue) selectivity filter structures. Apparent carbonyl displacements are given in Å.

Cryo-EM of Kv1.2s in Na+. (A) Size-exclusion chromatogram. Detector drift was large compared to a small protein signal. (B) Representative micrograph showing monodisperse particles on the graphene substrate. (C) Representative 2D classes. (D) Cryo-EM data processing workflow. (E) Gold standard FSC resolution estimation. (F) Local resolution estimation.

Comparisons of cryo-EM density map and model for alpha helices in each Kv1.2 structure reported here.

Cryo-EM data collection, refinement and validation statistics

Representative micrograph of Kv1.2 W366F in Na+, demonstrating the absence of protein aggregates on the graphene substrate.

Structural comparison of inactivated Kv channels.

(A-J) Structural superposition of Kv1.2s-W366F pore domain with other inactivated channels: side view with Shaker W434F (A) or Kv1.2-2.1-3m (F). Loop 1 conformational differences with Shaker W434F side view (B), top view (C); or with Kv1.2-2.1-3m side view (G), top view (H). Loop 2 conformational changes with Shaker W434F side view (D), top view (E); Kv1.2-2.1 3m side view (I), top view (J). (K) Table lists of the differences among the inactivated Kv channels. (L) H-bonds among the inactivated Kv channels. Adjacent subunits are shown as different colors, and a dashed black line denotes the subunit boundary. H-bonding patterns affect the stability of the inactivated state and an obvious difference is at the location 32’, where Shaker has Thr and Kv1.2 has Val. The mutation V32’T in the Kv1.2-2.1 background provides a new hydrogen bond that stabilizes the important residue D30’ in the inactivated conformation; this can be seen in the Kv1.2-2.1-3m structure (L, panel 3). Our inactivated Kv1.2s structure (containing V32’) nevertheless shows an H-bond network that includes Y28’ and D30’ along with main-chain atoms, possibly yielding a similar stabilization of the inactivated state (L, panel 1). The inactivated Shaker structure lacks H-bond partners for either Y28’ or D30’, which instead are exposed to solvent; however another H-bond network stabilizes the P-loop-S6 linker (L panel 2).

Summary of conformational changes in Kv channel inactivation. Upper panels: (A) Kv1.2 WT pore domain (PD) in green (B) Kv1.3 H451N PD in orange and (C) Kv1.2 W366F PD in orchid represent the relaxed, partially twisted and fully twisted P-loop respectively. Lower panels: cartoon illustration of (A) relaxed, (B) partially twisted and (C) fully twisted selectivity filter P-loop of Kv channels. D30’ and Y28’ residue side chains are shown as red and green ovals.