Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of mefenamic acid for fully saturated IKs channel complexes. A statistical difference in V1/2 compared to control is shown as p-value, determined using an unpaired t-test. NS denotes not significant. SEM is denoted by ±.

K41C-KCNE1 mutants prevent the agonist effect of mefenamic acid.

(A) Current traces of WT EQ (top) and K41C-EQ (bottom) in the presence of 100 µM mefenamic acid (Mef). A 4 s protocol was used with pulses from -150 mV or higher to +100 mV, in 10 mV steps, followed by a repolarization step to -40 mV for 1 s. Holding potential and interpulse interval were -80 mV and 15 s, respectively.

(B) G-V plots obtained from WT EQ tail currents (triangles) and K41C-EQ (circles) in the absence (control: black) and presence of Mef (100 µM: grey; 1 mM: light blue). Boltzmann fits were: WT EQ control (n = 6): V1/2 = 25.4 mV, k = 19.4 mV; WT EQ 100 μM Mef (n = 3): V1/2 = -80.3 mV, k = 41.3 mV; K41C-EQ control (n = 4): V1/2 = 15.2 mV, k = 18.4 mV; K41C-EQ 100 μM Mef (n = 4): V1/2 = 11.4 mV, k = 19.4 mV; and K41C-EQ 1 mM Mef (n = 3): V1/2 = 16.7 mV, k = 19.8 mV.

(C) Summary plot of V1/2 change (ΔV1/2) for WT EQ in the presence of 100 µM mefenamic acid, WT EQ vs K41C-EQ in control and K41C-EQ in the presence of 100 µM and 1 mM mefenamic acid. **** denotes a significant ΔV1/2 compared to control where p<0.0001.

MD prediction of mefenamic acid binding site in the ps-IKs model.

(A) Pseudo-KCNE1 (ps-KCNE1) used to predict Mef binding site. Extracellular residues of KCNE1 (top), ps-KCNE1 (middle) and KCNE3 (bottom). Below, cartoon topology of the single transmembrane ps-KCNE1 β-subunit and the six transmembrane KCNQ1 α-subunit. S1-S4 transmembrane segments form the voltage sensor domain and S5-S6 form the pore domain.

(B) Predicted binding pose of Mef (green) in external region of the ps-IKs channel complex (side view). The residues of the pore domain are coloured in blue, ps-KCNE1 subunit in red, and the voltage-sensor domain of a neighbouring subunit is presented in yellow.

(C) Mef binding pocket in space-fill formed by external S1 (yellow) and S6 (blue) transmembrane domains of KCNQ1 and extracellular region of the ps-KCNE1 subunit (red).

(D) Ligand interaction map of Mef with ps-IKs. Size of residue ellipse is proportional to the strength of the contact. The distance between the residue label and ligand represents proximity. Grey parabolas represent accessible surface for large areas. The 2D diagram was generated by ICM pro software with cut-off values for hydrophobic contacts of 4.5 Å and hydrogen bond strength of 0.8. Further details in Methods.

Current waveform and G-V changes induced by mefenamic acid in binding site mutants.

(A) Current traces from WT EQ and key residue mutants in control (black) and 100 µM Mef (colors).

(B) EQ-W323A and (C) EQ-Y148C current traces in control (top) and presence of 100 μM Mef (below). G-V plots in control (black) and presence of 100 μM Mef (colors). Boltzmann fits were: EQ-W323A control (n = 4): V1/2 = 47.8 mV, k = 23.7 mV; EQ-W323A Mef (n = 3): V1/2 = 33.7 mV, k = 28.5 mV; EQ-Y148C control (n = 4): V1/2 = 36.8 mV, k = 20.3 mV; and EQ-Y148C Mef (n = 4): V1/2 = 17.5 mV, k = 36.7 mV). Voltage steps were from -80 mV to +100 mV for 4 s, followed by a 1s repolarization to -40 mV. Interpulse interval was 15 s.

(D) Summary plot of the normalized response to 100 µM Mef (see methods). ** and * denote a significantly reduced response compared to WT EQ. N.S. denotes not significant.

(E) Plot of V1/2 response (ΔV1/2) in drug for different mutants compared with WT. Significance of differences as stated in Methods. n-values for mutants in D and E are stated in Table 1.

Mean V1/2 of activation (mV) and slope values (k-factor, mV) for WT EQ treated with 100 µM mefenamic acid, and untreated mutant EQ-L142C. Interpulse interval used is as indicated. SEM is denoted by ±. An interpulse interval of 7 s created such a dramatic change in the shape of the EQ-L142C G-V plot (see Figure 3-figure supplement 1) that a Boltzmann curve could not be fit and V1/2 and k-values are therefore not available.

K41C, Y46C and W323A mutant Impact on mefenamic acid binding energy and flexibility of external KCNE1 residues.

(A) Average free interaction energy of Mef-bound ps-IKs complexes calculated according to MM/GBSA method from three independent MD simulation runs. For K41C and W323A mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. * and *** denote significant differences in average free interaction energy compared to WT.

(B) Surface representation of WT ps-IKs after removal of Mef. The pore is blue, ps-KCNE1 is red and the VSD of a neighbouring subunit is yellow.

(C-D) Root mean square fluctuations (RMSF) of ps-KCNE residues (Å) in the ps-Iks complex during the last 100 ns of simulations. Three separate MD simulations shown for WT ps-IKs channel without Mef (black lines) and three for K41C (C, red) and W323A (D, blue) after ligand detachment. GROMACS software was used for RMSF analysis.

Average free interaction energies of MEF-bound ps-IKs complexes calculated according to MM/GBSA methods from three independent MD simulation runs. Mean values are in kcal/mol, ± SD. For W323A and K41C mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. Note that unbinding occurred in K41C and W323A in all 3 simulations, e.g. Supplemental movies S2 and S3.

Effect of DIDS on IKs.

(A) Molecular structure of mefenamic acid and DIDS.

(B) WT EQ current in control (black) and exposed to 100 µM DIDS over time (grey). Pulses were from -80 to +60 mV every 15 s, and current traces are shown superimposed. Lower panel shows no effect on currents from GFP-only transfected cell exposed to 100 µM DIDS over time (grey).

(C) Current traces from WT EQ in control and presence of 100 µM DIDS as indicated. Pulses were from -80 to +100 mV for 4s, with a 1 s repolarization to -40 mV. Interpulse interval was 15 s.

(D) Corresponding G-V plot in control (black) and DIDS (grey) from data as shown in panel C. Boltzmann fits were: WT EQ control (n = 8): V1/2 = 30.5 mV, k = 20.3 mV; WT EQ in the presence of DIDS (n = 5): V1/2 = -16.1 mV, k = 25.3 mV.

Mean V1/2 of activation (mV) and slope values (k-factor, mV) in the absence and presence of 100 µM DIDS for fully saturated IKs channel complexes. A statistical difference in V1/2 compared to control is shown as p-value determined using an unpaired t-test. NS denotes not significant. SEM is denoted by ±

Ligand interaction and energy decomposition per amino acid for DIDS binding to ps-Iks.

(A) Ligand interaction map of DIDS with ps-IKs. Size of residue ellipse is proportional to the strength of the contact. The distance between the residue label and ligand represents proximity. Grey parabolas represent accessible surface for large areas. The 2D diagram was generated by ICM pro software with a cut-off value for hydrophobic contacts 4.5 Å and hydrogen bond strength 0.8.

(B) Energy decomposition per amino acid for DIDS binding to ps-IKs. Generalized Born Surface Area (GBSA; orange) and Poisson-Boltzmann Surface Area (PBSA; blue) methods were used to estimate the interaction free energy contribution of each residue in the DIDS-bound ps-IKs complex. The lowest interaction free energy for residues in ps-KCNE1 and selected KCNQ1 domains are shown as enlarged panels (n=3 for each point).

Average free interaction energies of DIDS-bound ps-IKs complexes calculated according to MM/PBSA and MM/GBSA methods from three independent MD simulation runs. Mean values are in kcal/mol, ± SD. For W323A and Y46C mutations, calculations correspond to interval of simulations before the detachment of ligand from molecular complex. Note that unbinding occurred in W323A in 2 of 3 simulations, and K41C did not unbind but the binding pose shifted – see Supplemental movie S6.

Binding site mutants important for the action of DIDS.

(A) Current traces from WT EQ and key mutants in the absence (control; black) and presence (colors) of 100 µM DIDS. All calibration bars denote 0.5 nA and 0.5 s.

(B) Data and G-V plots in control (black) and DIDS (teal). Boltzmann fits were: K41C-EQ control (n = 3): V1/2 = 23.1 mV, k = 20.2 mV; K41C-EQ DIDS (n = 5): V1/2 = -1.6 mV, k = 24.0 mV. Voltage steps from a holding potential of -80 mV to +70 mV for 4 s, followed by repolarization to -40 mV for 1 s. Interpulse interval was 15 s. All calibration bars denote 0.5 nA and 0.5 s.

(C) Summary plot of the normalized response to 100 µM DIDS. For calculation, see Methods.

(D) V1/2 response (ΔV1/2) to 100 µM DIDS in different mutants compared to WT. Voltage protocol was as in panel B. n-values for mutants in C and D are stated in Table 4.

Average free energy of mefenamic acid and DIDS bound WT and mutant ps-IKs complexes.

Values are calculated according to the MM/GBSA method from three independent MD simulation runs and further broken down by residue and channel region. For K41C and W323C, calculations correspond to the interval of simulations before detachment of the ligand from the complex. Values are in kcal/mol. Mutated residues are in bold italics.

Subtle differences in activator binding to Y46C- and A44C-ps-IKs mutant channels.

(A) Mefenamic acid bound to the WT ps-IKs channel. Residues that are part of the binding site are shown in stick format colored green, except those residues that were important in the WT channel that had reduced ΔG in Y46C (in grey). Residues that had slight increases in ΔG values in Y46C are shown in magenta (S298, A300). Mefenamic acid is in cyan.

(B) DIDS bound to the WT ps-IKs channel. Residues that are part of the binding site are shown in stick format colored green except those residues that were important in the WT channel that had reduced ΔG in the A44C mutant (in grey). Residues that had slight increases in ΔG values in A44C are shown in magenta. DIDS is in cyan. Images were made with the PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.

Drug docking and MD simulation workflow.

(A) Schematic representation of the general workflow for construction of ps-IKs, Mef docking to the model ps-IKs channel complex and MD simulations (See Methods).

Energy decomposition per amino acid for mefenamic acid binding to ps-IKs.

Poisson-Boltzmann Surface Area (PBSA; blue) and Generalized Born Surface Area (GBSA; orange) methods were used to estimate the interaction free energy contribution of each residue in the Mef-bound ps-IKs complex. The lowest interaction free energy for residues in ps-KCNE1 and selected KCNQ1 domains are shown as enlarged panels (n=3 for each point).

Augmented activation of EQ-L142C in the absence of mefenamic acid.

(A) EQ-L142C current traces in control solutions. Voltage steps were from -130 mV or higher in 10 mV steps to +100 mV for 4 s, followed by a repolarization step to -40 mV for 1 s. Holding potential was -80 mV. Interpulse interval was as indicated above, 15 s, 30 s and 7 s.

(B) G-V plot of WT EQ in control (interpulse interval 15 s: black) and in the presence of 100 µM Mef (interpulse interval 15 s: grey closed circle; 30 s: grey open circle) and EQ-L142C in control solutions (interpulse interval 7 s: red; 15 s: blue; 30 s: green). Boltzmann fits were: WT EQ 15 s control (n = 6): V1/2 = 25.4 mV, k = 19.4 mV; WT EQ 15 s Mef (n = 3): V1/2 = -80.3 mV, k = 41.3 mV; WT EQ 30 s Mef (n = 8); V1/2 = 26.7 mV, k = 66.6 mV; EQ-L142C 15 s control (n = 6) V1/2 = -80.3 mV, k = 30.0 mV; EQ-L142C 30 s control (n = 3) V1/2 = -28.7 mV, k = 62.5. Due to the dramatic change in G-V plot shape, Boltzmann fits were not possible for EQ-L142C with an interpulse interval of 7 s. All mutations are forced saturated IKs channel complexes.

G-V plots for Y46A-EQ in the presence and absence of 100 mM mefenamic acid and DIDS.

G-V plots obtained from Y46A-EQ tail currents (circles and triangles) in the absence (control: black) and presence of Mef (100 µM: orange) and DIDS (100 µM: green).

Boltzmann fits were: Y46A-EQ control (n = 3): V1/2 = 76.4 mV, k = 57.3 mV; Y46A-EQ Mef (n = 3): V1/2 = -29.8 mV, k = 18.9 mV; Y46A-EQ control (n = 5): V1/2 = 75.2 mV, k = 59.1 mV; Y46A-EQ DIDS (n = 5): V1/2 = 25.8 mV, k = 39.2 mV.

MD simulations at the molecular level of binding of mefenamic Acid to WT EQ, and K41C-EQ,W323A-EQ, Y46C-EQ mutants. Note that videos are longer than the actual simulations, durations stated below.

Movie S1. 80 ns simulation of mef binding to WT ps-KCNE1-KCNQ1.

Movie S2. 150 ns simulation of mef binding to K41C ps-KCNE1-KCNQ1

Movie S3. 110 ns simulation of mef binding toW323A ps-KCNE1-KCNQ1.

Movie S4. 120 ns simulation of mef binding to Y46C ps-KCNE1-KCNQ1

MD simulations at the molecular level of binding of DIDS to WT EQ, K41C-EQ, andW323A-EQ.

Movie S5. 220 ns simulation of DIDS binding to WT ps-KCNE1-KCNQ1.

Movie S6. 250 ns simulation of DIDS binding to K41C ps-KCNE1-KCNQ1

Movie S7. 250 ns simulation of DIDS binding toW323A ps-KCNE1-KCNQ1.