Open Kv1.2 overall structure.

(A) Side and top view of the native Kv1.2 cryo-EM density map (upper panel) and model (lower panel). Lipid densities are colored gray in the map. (B) Side view of VSD structure and map density of Kv1.2 (yellow). (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 Kv1.2 WT (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 conformation.

(A) Sequence alignment of potassium channels in the S5-S6 linker region, with the relative numbering by Miller (1990) indicated at top. (B) Side view of opposing subunits in the open Kv1.2 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 Kv1.2 P-loops. On the left is shown the Kv1.2 residue numbering, and on the right the Miller numbering. (D) Side view of the inactivated Kv1.2 W17’F pore domain. The new locations of Y28’, F17’ and D30’ are shown in the right panels. (E) Side view of the Kv1.2 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) Top view of the Kv1.2 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 cantaloupe, respectively.

Kv1.2 DTX bound structure.

(A) DTX model and electrostatic surface view. Positively-charged sidechains are shown, and the three disulfide bridges are shown in yellow. (B) Representative 2D classes of the DTX-bound Kv1.2 particles. (C) The Kv1.2-DTX structure is shown as electrostatic surface view with DTX cryo-EM density (gray) visible at top. (D) Top view of the Kv1.2 DTX structure is shown as electrostatic surface view with (right panel) and without (left panel) DTX cryo-EM density (gray). (E) Side view of the Kv1.2-DTX selectivity filter. Ion density is marked as a blue ball. (F) Superposition of Kv1.2 DTX (colored) and Kv1.2 WT (gray) selectivity filter structures. Orange dashed lines show the distances between carbonyl groups of the Kv1.2 DTX structure. The corresponding distances in the absence of toxin are 5.0, 4.7, 4.7,4.7 and 5.5Å.

Summary of Kv1.2 conductive and non-conductive pores.

Selectivity filter structures of (A) Kv1.2 WT, (B) Kv1.2 WT Na+ bound, (C) Kv1.2 W366F, and (D) Kv1.2 DTX bound. Potassium ions and sodium ions are shown as blue and orange balls, respectively. Circled numbers label the P-loop carbonyl oxygens.

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. (E) Overall TMD map density fits with Kv1.2 W366F model. (F) Pore domain density fitting shows the absence of pore-loop density.

Structural comparison of inactivated Kv channels.

(A) Sequence alignment of potassium channels under Miller’s numbering system. Side view structural superposition of Kv1.2 W366F and Shaker W434F (B), Kv1.2-2.1 3m (G) pore domains. Loop 1 conformational changes between Kv1.2 W366F and Shaker W434F side view (C), top view (D); Kv1.2-2.1 3m side view (H), top view (I). Loop 2 conformational changes between Kv1.2 W366F and Shaker W434F side view (E), top view (F); Kv1.2-2.1 3m side view (J), top view (K). (L) H-bonds among the inactivated Kv channels, adjacent subunits are shown as different color with a dashed line. (M) Table lists of the differences among the inactivated Kv channels.

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

Selectivity filter structures and P- loop sequences of potassium selective non-conducting (A) Kv1.2 W366F in purple, (B) Kv1.3 alternate conformation in pink, (C) Kv1.3 H451N in orange, (D) Kv1.2-2.1 V406W in green. E,F Pore regions of less-selective channels, with corresponding pore sequences: the non-selective, conducting NaK in dark purple (E) and the weakly-selective, conducting HCN in light green (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 native Kv1.2.

(A) Representative micrograph. (scale bar 20 nm) (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 **low K** 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. (scale bar 20 nm) (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 Kv1.2 in (A) inactivated state (purple). (B) Kv1.2 VSD R3/E183 (upper), R4/E226 (middle), and K5/F233 (lower) interactions in the inactivated state. VSD structure in open state (yellow). (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) Kv1.2 (yellow) and Kv1.2-2.1 (light blue, PDB: 2R9R); (J) Kv1.2 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).

Cryo-EM of Kv1.2 DTX.

(A) Representative micrograph. (scale bar 20 nm) (C) Representative 2D classes. (C) Cryo-EM data processing workflow. (D) Gold standard FSC resolution estimation. (F) Local resolution estimation.

Structural comparison of Kv1.2 DTX bound structure with Kv1.2-2.1 CTX bound structure.

(A) Side view of the selectivity filter of Kv1.2-2.1 CTX bound conformation. Orange dashed lines show the distances between carbonyl oxygens, Potassium ions are shown as blue balls. (B) Superposition of Kv1.2 DTX (red) and Kv1.2-2.1 CTX (blue) selectivity filter structures. Carbonyl displacements are given in Å.

Cryo-EM of Kv1.2 in Na+.

(A) Representative micrograph. (scale bar 20 nm) (C) Representative 2D classes. (C) Cryo-EM data processing workflow. (D) FSC resolution estimation. (F) Local resolution estimation.

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

Conformational changes of Kv channel inactivation.

(A) Kv1.2 WT 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. 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.