Design and characterization of SK2-4 chimera.

A) Cartoon representation of the SK2-4 chimera. Residue numbers in the SK2 regions of the structure correspond to the human SK2 sequence described by Desai et al.31 and the SK4 regions of the structure are numbered sequentially from the SK2 regions. The sequence alignment of human SK2 (green) and human SK4 (cyan) indicate the chimera boundaries (black lines). B) Representative current traces in response to a ramp voltage protocol from -120 mV to 80 mV show that reversal potentials of WT SK2 (black), SK4 (blue), and SK2-4 (green) currents are all around -85 mV demonstrating similar potassium selectivity.

Shown SK current traces are isolated by subtracting leak current under saturating concentration of inhibitors (UCL1684 or TRAM-34). C) SK2-4 (green squares) is activated by increasing intracellular Ca2+ like WT SK2 (black circles) and SK4 channels (blue triangles). Current amplitudes were normalized to average value at 20 µM Ca2+. Apparent EC50s for SK2, SK4 and SK2-4 are 5.4 µM, 0.8 µM and 0.4 µM, respectively. Note that actual intracellular free [Ca2+] might differ from intracellular buffer due to limit of diffusion in whole-cell configuration. Data points reflect mean +/- SEM (n=4) D) SK2-4 is inhibited by apamin (blue circles, IC50=0.7 nM), which targets the extracellular domains of SK2, and AP14145 (black squares, IC50=2.4 μM), which targets the transmembrane domains of SK2. Data points reflect mean +/- SD (n=4).

SK2-4 chimera architecture.

Structures of Ca2+-bound (A) and Ca2+-free (B) SK2-4. SK2-4 is shown as a cartoon with each subunit of the tetramer a different color. The grey lines (left) indicate membrane boundaries. In Ca2+-bound SK2-4 (A) both the N- and C-lobes of CaM (purple surface) are associated with the intracellular domains. In Ca2+-free SK2-4 (B) only the CaM C-lobe is bound (purple surface) and the N-lobe is dissociated (not shown). C) Overlay of the K+ pore from Ca2+-bound (green) and Ca2+- free (lavender) SK2-4 structures. Same view as left panels of (A) and (B) but only 2 subunits are shown. D) Pore radii of Ca2+-bound (green), Ca2+-free (lavender), and apamin-bound (orange) SK2-4. The location of the intracellular gate (Val390), selectivity filter (SF), and extracellular constriction (Phe243) are indicated. Grey dashed line indicates the radius of hydrated K+ (3 Å).

S3-S4 loop architecture.

A) Extracellular view of Ca2+-bound SK2-4 with each subunit of the tetramer a different color. The S3-S4 linker (surface and cartoon) extends over the S5 and S6 helices. Phe243 residues (spheres) form an extracellular constriction with a radius of 1.8 Å. Boxes indicate location of the interactions shown in (B) and (C).

B) Interactions between the S3-S4 linker and the C-terminus of S5. His336 forms an edge-to-face interaction with Trp237 and hydrogen bond with Ser248 (dashed lines). Tyr335 forms a hydrogen bond with D253 (dashed lines). C) Interactions between the S3-S4 linker and the C-terminus of the selectivity filter. Arg240 and Tyr245 (green sticks) from the S3-S4 linker form a salt bridge and hydrogen bond (dashed lines) with side chain and backbone carbonyl of Asp363 from the neighboring subunit (orange sticks), respectively. Phe243 (green sticks) forms an edge-to-face interaction with the neighboring Phe243 (orange sticks) and is in position to form a C-H/O interaction (dashed line) with Gly262 (green sticks) from the same subunit.

Selectivity filter conformation of SK2-4.

A) Ca2+-bound SK2-4 selectivity filter structure (green). Arg240 and Tyr245 from the S3-S4 form a salt bridge and hydrogen bond (dashed lines) with Asp363 and the selectivity filter adopts a conformation with two K+ coordination sites (purple spheres, density shown). B) Sequence alignment of selectivity filter from K+ selective channels (hSK2, hSK4, KcsA, hEag) and non-selective cation channels (hHCN and NaK) C) Structure of hSK4 selectivity filter (cyan, PDB: 6CNN). In SK4 there is a conserved hydrogen bond between the selectivity filter Asp255 and pore helix Trp242 (dashed lines) and the selectivity filter conformation creates four occupied K+ coordination sites (purple spheres). D) Overlay of the SK2-4 (green) and the SK4 (cyan) selectivity filter with a 90° rotation from (C). E) Structure of NaK selectivity filter (magenta, PDB: 2AHZ). In NaK there is no interaction between the selectivity filter Asn68 and the pore helix Tyr55 and the selectivity filter adopts a conformation with two occupied K+ coordination sites (purple spheres). F) Overlay of the SK2-4 (green) and the NaK (magenta) selectivity filter with a 90° rotation from (E).

Mechanism of apamin inhibition.

A) Extracellular view of apamin binding site. Apamin (cyan ribbon, density shown) binds to the S3-S4 extracellular gate. Apamin Arg13 and Arg14 (cyan sticks) and S3-S4 linker residues (orange) that surround the apamin binding site are shown as sticks. B) Apamin sensitivity of WT SK2 (black squares) and SK2 F243A (blue circles). Data points represent mean +/- SD (n=6). The F243A mutant is insensitive to apamin up to 3 µM. C) Overlay of Ca2+-bound SK2-4 selectivity filter (green cartoon) and apamin-bound SK2-4 selectivity filter (orange, selectivity filter shown as sticks and S3-S4 linker and pore helix shown as a cartoon).

Upon apamin (cyan cartoon) binding the S3-S4 linker retracts from the pore axis (black arrow) and the selectivity filter adopts a conformation with four K+ coordination sites (purple spheres, density shown). D) 90° degree rotation of (C).

Mechanism of compound 1 inhibition.

A) Structure of compound 1 (inhibitor) and compound 4 (activator). B) Potency of compound 1 on SK2 (black circles, IC50=69 nM), SK2-4 (green squares, IC50=140 nM), and SK4 (blue triangles, IC50=0.66 µM). Data points represent mean +/- SD (n=4). C) Melting curves of Ca2+-bound SK2-4 in the absence (green, Tm = 58.7 °C) and presence (yellow, Tm = 69.5 °C) of compound 1 measured by CPM indicates target engagement. D) Compound 1 (magenta sticks, density shown) interacts with a pocket formed by the S5, pore helix, and S6. Ser318 of S5 is in position to hydrogen bond (dashed line) with the sulfonamide nitrogen of compound 1. E) Overlay of the K+ pore of Ca2+-bound SK2-4 (green) and compound 1- bound SK2-4 (yellow) (only 2 subunits are shown for clarity). The methoxy of compound 1 (magenta spheres) clashes with S6 Thr386 (spheres) and induces a movement of the S6 and S45B helices toward the pore axis (arrow) to close the intracellular gate (Val390, spheres). D) Pore radii of Ca2+-bound (green), compound 1-bound (yellow), and compound 4-bound (grey) SK2-4. The location of the intracellular gate (Val390), selectivity filter (SF), and extracellular constriction (Phe243) are indicated. Grey dashed line indicates the radius of hydrated K+ (3 Å).

Mechanism of compound 4 activation.

A) Comparison of activation and inhibition curves for compound 4 (orange squares) and compound 1 (magenta squares), respectively. Data points represent mean +/- SD (n=6). B) Compound 4 (orange sticks, density shown) interacts with a pocket formed by the S5, pore helix, and S6 and the trifluoromethyl extends towards Ile380 on the neighboring S6 (S6’). C) Overlay of compound 1-bound SK2-4 (yellow) and compound 4-bound (grey) SK2-4. In the closed state of the S6 helices (yellow cartoon) the trifluoromethyl of compound 4 clashes with Ile380 (yellow sticks) on the S6 helix (S6’) of the neighboring subunit (2.3 Å distance). In the open state of the S6 helices (grey cartoon), Ile380 (grey sticks) is 3.1 Å distant the trifluoromethyl minimizing this clash.

Sequence alignment of human SK1, SK2, SK3, and SK4.

SK2 structural features and chimera boundaries are annotated. Sequence identity is shown in the graph above the sequences.

Structure determination of Ca2+-bound SK2-4 and Ca2+-free SK2-4.

A) 2D class averages of WT SK2 showing disorder in the intracellular region. Cryo-EM structure determination flowchart for (B) Ca2+-bound SK2-4 and (C) Ca2+-free SK2-4. (D) FSC curves, (E) angular distribution, and (F) local resolution estimates for Ca2+-bound SK2-4 and Ca2+-free SK2-4. G) Interactions between the SK2-4 intracellular domains and CaM (surface, purple) in the Ca2+-bound conformation of SK2-4 (cartoon, each subunit has a different color). The CaM C-lobe interacts with the HA and HB helices and the N-lobe interacts with the S45A. H) Overlay of the intracellular domains from SK2-4 (colored as in G) and SK4 (grey) demonstrate a similar CaM binding interaction in each structure.

Representative cryo-EM densities.

Cryo-EM densities of the S3, S3-S4 linker, S4, S45B, S5, pore helix, and S6 for (A) Ca2+-bound SK2-4, (B) Ca2+-free SK2-4, (C) apamin-bound SK2-4, and (D) compound 1-bound SK2-4.

Cryo-EM density for the SK2-4 selectivity filter.

Cryo-EM density for the selectivity filter and interacting S3-S4 residues (not shown in 90° rotated view) for (A) Ca2+-bound SK2-4, (B) Ca2+-free SK2-4, (C) apamin-bound SK2-4, and (D) compound 1- bound SK2-4.

Structure determination of apamin-bound SK2-4 and compound 1- bound SK2-4.

Cryo-EM structure determination flowchart for (A) apamin-bound SK2-4 and (B) compound 1-bound SK2-4. (C) FSC curves, (D) angular distribution, and (E) local resolution estimates for apamin-bound SK2-4 and compound 1-bound SK2-4.

Characterization of compound 1 inhibition.

A) Potency of compound 1 on WT SK2 (black squares, IC50=69 nM), hNav1.5 (cyan squares, IC50=15.3 µM), hERG (red circles, IC50=13 µM), hCav1.2 (blue triangles, IC50 >50 µM), and hKCNQ1 channels (purple stars, IC50 >50 µM). Data points represent mean +/- SD (n=4-6). B) Overlay of the compound 1 (magenta sticks) binding site from the structures of compound 1-bound SK2-4 (yellow) and Ca2+-bound SK2-4 (green). A rotation of Leu321 is required to accommodate the benzoxadiazole. The methoxy of compound 1 (magenta sticks) clashes with the S6 Thr386 in Ca2+-bound SK2-4. Surface representation of the compound 1 binding pocket in (C) compound 1-bound SK2-4 (yellow cartoon and grey pocket) and (D) Ca2+-bound SK2-4 (green cartoon and grey pocket). Rotation of Leu321 is required to expand the pocket and accommodate the benzoxadiazole of compound 1. E) Overlay of Ca2+-bound SK2-4 selectivity filter (green cartoon) and compound 1-bound SK2-4 selectivity filter (yellow, selectivity filter shown as sticks and S3-S4 linker and pore helix shown as cartoons). Upon compound 1 binding the S3-S4 linker moves away from the pore axis (black arrow) and the selectivity filter adopts a conformation with four K+ coordination sites (purple spheres, density shown). F) 90° degree rotation of (E). G) Overlay of Ca2+-free SK2-4 (lavender) and compound 1-bound SK2-4 (yellow). The S6 and S45B helices adopt a similar conformation in the Ca2+-free and compound 1 (magenta spheres)-bound states with a closed intracellular gate (Val390, spheres). H) Structure of compound 2 and compound 3. I) Removal of the methoxy from compound 2 (black squares, IC50=0.51 µM) produces compound 3 (blue circles, IC50=18 µM) with a 30-fold reduction in potency. Data points represent mean +/- SD (n=6).

Structure determination of compound 4-bound SK2-4.

Cryo-EM structure determination flowchart (A), FSC curves (B), angular distribution (C), and local resolution estimates (D) for compound 4-bound SK2-4. E) Cryo-EM densities of the S3, S3-S4 linker, S4, S45B, S5, pore helix, and S6 for compound 4-bound SK2-4. D) Cryo-EM density for the selectivity filter and interacting S3-S4 residues (not shown in 90° rotated view) for compound 4-bound SK2-4. H) Overlay of compound 1 (magenta sticks) bound to SK2-4 (yellow cartoon and sticks) and compound 4 (orange sticks) bound to SK2-4 (grey cartoon and sticks).

Data collection parameters and Refinement statistics