Structural and kinetic mechanisms of state-dependent potassium channel inhibition

Figures

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  Fig. 1.. Structures of the xWT activated-state (PDB ID: 9MY3) and xE1R/R2E intermediate-state (PDB ID: 9MY4) KCNQ1 channels. (A) Side and top views of the xWT activated-state structure. One pair of domain-swapped protomers is colored purple and yellow, and the second pair is shown in white for clarity. CaM is displayed as tube helices. (B and C) VSD configurations of the xWT-activated (B, purple) and xE1R/R2E-intermediate (C, green) structures (20). Positively charged residues are shown in blue [R1 (xR218), R2 (xR221), R4 (xR227), H5 (xH230)], negatively charged residues in red [E1 (xE150)], and Q3 (xQ224) in pink. The adjacent FH helix (FH′) is highlighted in yellow to emphasize its spatial relationship with S1 in each state. (D) 2D chemical structure of UCL2077. (E) Whole-cell patch recordings of xE1R/R2E exposed to UCL2077. Construct as used for cryo-EM, but uncleaved from GFP, expressed in tsA201 cells. Pulses were every 15 s from a holding potential of −90 to 60 mV for 4 s and then to −40 mV for 0.9 s to record tail currents. Only tail current and holding current are shown, before (blue) and after 5 pM (cyan) or 10 pM (green) UCL2077. The dashed line indicates 0 pA.

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  Fig. 2.. Cryo-EM maps of xE1R/R2E-CaM in the absence or presence of UCL2077. Diagonally opposed protomers of the xE1R/R2E channel in the presence or absence of UCL2077 are colored red/orange and green/lime, respectively. CaM densities are colored white, unmodelled densities dark gray, and UCL2077 densities yellow. (A) Side-view cryo-EM reconstruction of the xE1R/R2E-CaM-UCL2077 complex. 0° and 90° views are shown due to the C2 symmetry of the map. (B) Cryo-EM map of the native C4-symmetric xE1R/R2E-CaM channel for reference (20). Insets zoom in on the corresponding binding site region. (C and D) Top view cryo-EM map cross section at the level of UCL2077 for the ligand-bound (C) and native channels (20) (D). (E) UCL2077 fit in the cryo-EM map. The xE1R/R2E-UCL2077 cryo-map is shown at a contour level of 0.0275 and for native xE1R/R2E at 0.103.

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  Fig. 3.. Structure of the xE1R/R2E-CaM-UCL2077 complex and interactions with binding site residues. Diagonally opposed protomers of xE1R/R2E are colored red/orange, CaM in white, and UCL2077 in yellow. (A) Side view of the tetrameric channel showing the UCL2077 bound at the interface formed by the S5-S6 helices of one protomer and the S6 helix of the adjacent protomer. The inset provides a close-up of the ligand binding pocket, highlighting residues that interact with UCL2077 and S328P2, which does not. (B) Side view of the complex channel structure rotated 90° relative to (A) to reveal additional interacting residues not visible in the initial orientation. (C) Representative tail current traces of WT hKCNQ1 and the hI337V mutant in control (blue) 0.3 nM UCL2077 (green) or 1 μM HMR1556 (magenta). Cells were pulsed from a holding potential of −90 to 60 mV (except hF340, in (D), which was pulsed to +40 mV) for 4 s and then for 0.9 or 2 s at −40 mV. Dotted lines denote zero current. (D) Initial tail current amplitudes in 300 pM UCL2077 were normalized to control values before drug exposure. Bars represent means ± SEM. Only values that were statistically different from WT using one-way Anova…

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  Fig. 4.. MD simulations of the xE1R/R2E-UCL2077 complex structure confirm stable ligand binding across replicas. (A) Portion of the channel used for the 1 μs MD simulations. Only the PDs that bind UCL2077 (red) and their domain-swapped VSDs (orange) are shown for clarity. Graph shows RMSD over time, with kernel density estimates (KDEs) of aggregated replicas on the right. (B) Ligand binding dynamics in the two binding pockets over the course of the simulations. The initial positions of the binding site residues and the ligand are shown as red and yellow cylinders, respectively, with conformational changes during the simulation represented as gray sticks and captured every 50 ns. Graphs below show ligand RMSD in each pocket over time, with KDE of aggregated replicas on the right.

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  Fig. 5.. Conformational changes in the S6 helix upon UCL2077 binding in the xE1R/R2E structure. Native xE1R/R2E (20) is shown for reference in white, UCL2077-bound xE1R/R2E protomers in red (P1 and P3), UCL2077-free protomers in orange (P2 and P4), and UCL2077 in yellow. (A) The S6 helix adopts a helix-loop-helix configuration at the PAG motif in the UCL2077-bound protomers. xP333 (PAG motif) and xF330 are colored black. (B) Close-up view of the UCL2077 binding region, rotated 90° relative to (A), highlighting local S6 conformational changes associated with UCL2077 binding. (C) Side views showing distances between gate residues in the native xE1R/R2E and ligand-bound structure. (D) Pore-axis radius profiles for the native xE1R/R2E and UCL2077-bound structure with narrower regions shown in red and wider regions in blue. Only two diagonally opposed protomers are shown for clarity; inset shows a top-view representation emphasizing the restriction at the level of xF330. Gate residues (xG335, xS339, and xL343), xI327, and xF330 are shown as green, cyan, and orange cylinders, respectively. (E) HOLE pore radius profiles of the native (dashed black line) and drug-bound (in red) structures and the…

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  Fig. 6.. UCL2077 induces shifts in the VSD in the xE1R/R2E structure. Comparison of domain-swapped PD-VSD pairs in ligand-bound and ligand-free xE1R/R2E protomers. The xE1R/R2E native structure (20) is shown for reference in white, UCL2077 PD-bound protomers in red (P1 and P3), PD-free protomers in orange (P2 and P4), and UCL2077 in yellow. Top (A and B) and side (C and D) views are shown. (A and C) Comparison focused on the PD-bound VSD (P1, red) and the adjacent UCL2077-free PD (P2, orange). (B and D) Comparison focused on the UCL2077-bound PD (P1, red) and the adjacent PD-free VSD (P4, orange). In (C) and (D), the S6 helix diagonally opposed to the VSD has been removed for clarity. The plots show KDEs of distances between PDs (A290Cα) and VSDs (I135Cα) for the four protomers from aggregated MD replica data. Vertical lines indicate reference values in the cryo-EM structures.

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  Fig. 7.. The binding of UCL2077 in the xWT channel. Two side views rotated by 90° and a top view of the cryo-EM map (A) and structural model (B) of xWT in the presence of UCL2077. Insets show zoom-in of UCL2077 binding site. Diagonally opposed protomers that bind UCL2077 (P1 and P3) are colored blue and the protomers that do not bind UCL2077 (P2 and P4) are colored cyan. UCL2077 is colored yellow, CaM light gray, and unmodelled densities dark gray. (C) UCL2077 fit in the cryo-EM map. (D) Distance between α carbons of S6 gate residues in the native (20) (white; PDB-ID: 9MY3) and UCL2077-bound (blue/cyan) protomers. (E) Top view representation of xWT-UCL2077 (shades of blue) and xWT native (20) (white) clipped at the level of UCL2077 binding site. (F) Pore radius profiles for the native xWT and UCL2077-bound structures with narrower regions shown in red and wider regions in blue. Only two diagonally opposed protomers are shown for clarity; insets show top-view representations emphasizing the restriction at the level of xI327 and xF330. Gate residues (xG335, xS339, and xL343), xI327, and xF330 are shown as green, cyan, and orange cylinders, respectively. (G) Pore radius profiles of the…

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  Fig. 8.. The C2-symmetric binding of UCL2077 is favored in the intermediate state. (A to C) Top-view representation, clipped at the level of xF330, with diagonally opposed protomers colored red and orange. xI327 and xF330 are shown as cyan and green cylinders, respectively. (A) Structure of native xE1R/R2E (20) with four xF330 facing outside the inner vestibule. (B) xE1R/R2E-UCL2077 structure with two xF330 residues in the inner vestibule. (C) Tetramer generated by two copies of the UCL2077-bound protomers with four xF330 residues in the inner vestibule. Clashes are shown as purple and green dashed lines. (D) Side-chain orientation of xF330 in (left to right): xE1R/R2E bound, xWT-bound, xE1R/R2E-intermediate, and xWT-activated simulated systems, averaged over three replicates with SEM. Data from bound structures are distinguished by chains pairs (red and blue bars: pair P1/P3 in xE1R/R2E and xWT, respectively; orange and cyan bars: pair P2/P4 in xE1R/R2E and xWT, respectively). (E) MD-derived conformation of native xE1R/R2E-intermediate state with two diagonally opposed xF330 residues in the inner vestibule (flipped-in). The diagonally opposed protomer pairs are colored red and orange.…

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  Fig. 9.. UCL2077 selectivity for the xE1R/R2E-intermediate state of KCNQ1. (A) KCNQ inhibitor binding sites mapped on the PD of the xKCNQ1-UCL2077 structure. The UCL2077-bound and -free protomers are colored red and orange, respectively. UCL2077 is colored yellow, Ebio3 (30) (KCNQ2; PDB-ID: 9IXZ) magenta, and linopirdine (31) (LIN, KCNQ4, PDB-ID: 7BYN) cyan. Ligands are shown as ball and stick. (B to E), surface representation of the fenestration formed between the S5-S6′ helices at the putative UCL2077 binding sites, with UCL2077 shown as a translucent yellow surface in xE1R/R2E-UCL2077 (B), native xE1R/R2E (20) (C), native KCNQ2 (PDB ID: 7CR3) (67) (D), and native KCNQ4 PDB ID: 7BYL) (31) (E) structures. (F to K) Domain-swapped VSDP4-PDP1 and the diagonally opposed VSDP4-PDP2 packing in native xE1R/R2E PDB ID: 9MY4) (20) (F), xE1R/R2E-UCL2077 (G), native KCNQ2 PDB ID: 7CR3) (H), native KCNQ4 PDB ID: 7BYL) (I), native xWT PDB ID: 9MY3) (20) (J), and xWT-UCL2077 (K) structures. The backbone of the UCL2077 bound PDP1 and its adjacent UCL2077-free S1P4 (VSDP4) or S6P2 (PDP2) are shown in dark or light surface representations, respectively. Arrows show the shortest distance between the S1P1…