Mechanism of SK2 channel gating and its modulation by the bee toxin apamin and small molecules

Figure 1.. Design and characterization of the 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., 2000 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) indicates the chimera boundaries (black lines). (B) Representative current traces recorded with the Qube instrument in response to a ramp voltage protocol from –120 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. The shown SK current traces are isolated by subtracting leak current determined under saturating concentration of inhibitor (UCL1684 or TRAM-34). Intracellular solution contains 2 µM Ca2+ based on calculation using Maxchelator (see methods). (C) SK2–4 (green squares) is activated by increasing intracellular Ca2+ like WT SK2 (black circles) and SK4 channels (blue triangles). For each channel type, average SK current amplitudes from different cell populations each exposed to a different…

Figure 1—figure supplement 1.. 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.

Figure 2.. 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 in a different color. The gray 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) is indicated. Gray dashed line indicates the radius of hydrated K+ (3 Å).

Figure 2—figure supplement 1.. Structure determination of Ca2+-bound SK2–4/CaM and Ca2+-free SK2–4/CaM. (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 helix. (H) Overlay of the intracellular domains from SK2–4 (colored as in G) and SK4 (gray) demonstrates a similar CaM-binding interaction in each structure.

Figure 2—figure supplement 2.. Representative cryo-EM densities. Cryo-EM density shown at 5 σ 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.

Figure 3.. S3–S4 loop architecture. (A) Extracellular view of Ca2+-bound SK2–4 with each subunit of the tetramer in 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 the 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.

Figure 4.. Selectivity filter conformation of SK2–4. (A) Ca2+-bound SK2–4 selectivity filter structure (green). Arg240 and Tyr245 from the S3–S4 linker 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 at 5 σ). (B) Sequence alignment of selectivity filter from K+ selective channels (hSK2, hSK4, KcsA, and hKv1.3) 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), Tyr253 hydrogen bonds with Thr247, 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 90o rotation from (C). (E) Structure of HCN selectivity filter (magenta, PDB: 5U6O). In HCN, there is no interaction between the Ala363 at the C-terminus of the selectivity filter and the pore helix Phe350. Tyr361 is in a similar conformation to Ca2+-bound SK2–4, and the selectivity filter adopts a conformation with two ion…

Figure 4—figure supplement 1.. Cryo-EM density for the SK2–4 selectivity filter. Cryo-EM density shown at 5 σ 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.

Figure 5.. Mechanism of apamin inhibition. (A) Extracellular view of apamin-binding site. Apamin (cyan ribbon, density shown at 5 σ) binds to the S3–S4 extracellular gate. Apamin Arg13 and Arg14 (cyan) and the 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). Change in SK current amplitude for different cell populations each exposed to a different extracellular apamin concentration was normalized to DMSO control to construct dose responses. 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) 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) binding, the S3–S4 linker retracts from the pore axis (black arrow). Asp363 reorients into hydrogen-bonding position with a rotated Trp350 side chain, Tyr361 in the selectivity filter is rotated 180° to form a hydrogen bond with Thr355 in the pore helix, and the selectivity filter adopts a conformation with four K+ coordination…

Figure 5—figure supplement 1.. Structure determination of apamin-bound SK2–4/CaM and compound 1-bound SK2–4/CaM. 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.

Figure 6.. 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 = 660 nM). Change in SK current amplitude for different cell populations each exposed to a different extracellular compound 1 concentration was normalized to DMSO control to construct dose responses. 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. Dotted lines show individual replicates while solid lines show the nonlinear fit of all data for each condition. Raw data were analyzed by fitting melt curves using an extended Boltzmann function to determine melting temperature Tm for each replicate, and then taking the mean of the three replicates for each condition. Thermal stabilization value was determined by subtracting the mean Tm value of the apo samples from the mean Tm value of the compound 1 samples. (D) Compound 1 (magenta sticks, density shown at 5 σ) interacts with a pocket formed…

Figure 6—figure supplement 1.. 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). See Methods for detailed information regarding ion channel assays. (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. (C) Surface representation of the compound 1-binding pocket in compound 1-bound SK2–4 (left, yellow cartoon and gray pocket) and Ca2+-bound SK2–4 (right, green cartoon and gray pocket). Rotation of Leu321 is required to expand the pocket and accommodate the benzoxadiazole of compound 1. (D) Overlay of the compound 1 (magenta sticks)-binding site from the structures of compound 1-bound SK2–4 (yellow) and SK4 (cyan, PDB: 6CNM). Two changes in SK4 occlude the compound 1-binding site and may explain the…