PUFAs activate KCNQ1/KCNE1 channels.

A) (black) Prolonged QT interval in the ECG is due to for example loss-of-function mutations of KCNQ1/KCNE1 channels that generate the IKs current that normally contributes to the repolarizing phase of the ventricular AP. (red) PUFAs are potent activators of KCNQ1/KCNE1 channels that can restore the normal functioning of the channel and restore the AP duration and the QT interval. B) Representative current traces of KCNQ1/KCNE1 in 0 μM and 20 μM of Lin-Glycine. Voltage protocol on top. C) Conductance versus voltage curves from tail currents (measured at arrows in B). Channel activation by PUFA results in two main effects: a shift of the voltage-dependence of activation (ΔV0.5) and an increase in the channel maximum conductance (ΔGmax). D) KCNQ1 transmembrane topology. Residues mutated in this study are labeled. E) KCNQ1 top view (PDB: 6UZZ) with PUFA binding sites: Site I, at the VSD; and Site II, at the pore domain. The four subunits are shown in four different colors. F) Cartoon of PUFA mechanism of action. Site I, top panel. Electrostatic interactions between PUFA head groups and positively charged residues in S4 facilitate channel activation by stabilizing the outward state of S4. Site II, bottom panel. PUFA interaction with residues in the pore domain facilitates the increase in the maximum channel conductance.

Lin-Glycine induces an increase in the Po of KCNQ1/KCNE1.

A-B) 10 consecutive traces of KCNQ1/KCNE1 in A) control and B) in the presence of Lin-Glycine (20 μM) (top) Protocol used for the recordings. C-D. All-point amplitude histogram of 50 consecutive traces in C) control and D) Lin-Glycine). Note no change in the single-channel current amplitude, however an increase in the number of sweeps with channel opening is observed. Note that there were at least two channels in this patch. Different sweeps were assigned different colors to better visualize different types of channel behaviors. Note panels A-D are all from the same patch. E) Average currents of 100 sweeps in control and Lin-Glycine. F) All-point histogram of the last second of non-empty sweeps in control and in Lin-Glycine. We estimated the open probability from the all-point amplitude histogram by Po = Sum (iN/(iestimateNtotal)), where N is the number of points for a specific current i in the histogram, iestimate = 0.4 pA from the peak of the histogram, and Ntotal = 10,000 is the total number of points in the last second of the trace. p = 0.78 ± 0.02 (n = 8 sweeps) and p = 0.87 ± 0.04 (n = 8 sweeps) for Control and Lin-Glycine, respectively, from the same patch.

First latency to opening is shortened by Lin-Glycine.

A-B) Representative diary plot of first latency to opening during 4 s depolarizations to +60 mV for A) KCNQ1_WT /KCNE1 (EQ) and B) KCNQ1_Y315F/KCNE1 (Y315F) in control (left) and 20 μM Lin-Glycine (right). Latency values of zero correspond to sweeps with no channel openings. Green arrow indicates point at which Lin-Glycine (LG) was added. Mean ± SEM first latency for each cell before and after Lin-Glycine (LG) are indicated next to each plot. Note that these are representative traces and for some patches there are more recordings than shown. In the analysis in the main text we used only traces recorded clearly before application of Lin-Gly and after Lin-Gly had been applied for several minutes, and we used only patches with low number of openings to avoid complications of simultaneous channel openings if more than one ion channel was present in the patch. Note that left and right panels in each row are from the same patch, but each row is from a different patch.

Open probability stays high in KCNQ1/KCNE1 channels once opened.

A) 10 consecutive traces of KCNQ1/KCNE1in response to 20-sec long voltage steps in control solutions. (top) Protocol used for the recordings. B) Average currents of 57 sweeps in control solution.

PUFA binds to a state-dependent small crevice between K326 and D301.

A-B) KCNQ1_D301E/KCNE1 representative current traces A) in 0 μM of Lin-Glycine. B) After perfusion of 20 μM of Lin-Glycine. C) Gmax/Gmax0 for KCNQ1/KCNE1 channels and KCNQ1_D301E /KCNE1 channels. Gmax/Gmax0 was significantly reduced for the D301E mutation compared to WT channels (p = 0.0018, n = 4 oocytes). Student t test was used to conduct statistical analysis. D, E and F) Structures of crevice between S5 and S6 in KCNQ1 with S4 up (D and E) and S4 down (F). Residues that surround the crevice from S6 shown in blue (K326, T327, S330, V334) and from the pore helix and S5 in red (D301, A300, L303, F270). Remaining KCNQ1 residues shown in purple. On the left is shown the location of the crevice in KCNQ1 (top) side view and (bottom) top view. D) In MD simulations14, linoleic acid (LIN: gold color) fits in a narrow crevice present in the cryoEM structure of activated state KCNQ1 (S4 up). E) Same view as, in D but without LIN. F) In the cryoEM structure with S4 in the resting state (S4 down), the crevice between K326 and D301 is too narrow to fit LIN.

Different conformations of selectivity filter in cryoEM structures with S4 activated or resting.

A) Selectivity filter of KCNQ1 with S4 activated (S4 up; PDB: 8SIK). Distances between D317 and W305 and between T309 and Y315 are short enough to form hydrogen bonds (dashed lines). B) Selectivity filter of KCNQ1 with S4 in resting state (S4 down; PDB: 8SIN). Distances between D317 and W305 and between T309 and Y315 are too long for hydrogen bonds (dashed lines). Only two subunits are shown for clarity. C-D). Aromatic cuff in KCNQ1 with C) S4 activated and D) resting S4. Note how P320 (red) moves away from its position in between W304 and W305 from two different subunits in the S4 down conformation.

Effect of Lin-Glycine in shifting the voltage-dependence of activation (ΔV0.5).

A similar shift in the voltage dependence of activation was found for KCNQ1_D301E/KCNE1 and KCNQ1/KCNE1 after perfusion of several concentrations of Lin-Glycine. Comparisons at 20 μM of Lin-Glycine revealed no significant difference between the effect seen in KCNQ1/KCNE1 and KCNQ1_D301E (p = 0.52; n = 3 oocytes).

Atomic displacements at the selectivity filter and nearby regions in the transition from up to down state.

Each vector (shown in orange) starts from the position of a given atom in the up state (PDB ID 8SIK) and points to the position of the same atom in the down state (PDB ID 8SIN). A. Displacement of the Ca atoms of residues 298 to 341 is shown in orange if greater than 2 Å. The side chain of such residues is shown as sticks. B. Displacement of the side chains of residues 298 to 341. The side chain of residues W304, W305, T309, I313, Y315, D317, K318, V319, P320, K326 are highlighted as sticks and atoms are colored based on the atomic displacement values. Scale bar is in Å. In A and B, for clarity, only one monomer of the up state of KCNQ1 is shown.

Graphic indicating the atomic displacement of residues at the selectivity filter and nearby regions in the transition from up to down state.

Displacement of the Ca atoms of residues 298 to 341 is shown if greater than 2 Å. A) Displacement of atoms (Å) before the selectivity filter; B) residues of the selectivity filter; C) residues located just outside the selectivity filter region,

The ability of Lin-Glycine to increase the channel conductance is reduced when channel pore residues are mutated.

A) Gmax/Gmax0 values obtained for KCNQ1_WT/KCNE1 channel (black) and mutant channels. KCNQ1_Y315F/KCNE1 (red), KCNQ1_D317D/KCNE1 (blue), KCNQ1_T309S/KCNE1 (green) and KCNQ1_P320L/KCNE1 (purple), the Gmax/Gmax0 is significantly reduced. (p<0.0001(n = 4 oocytes). One-way ANOVA with Dunnett’s post-hoc multiple comparisons test was used for statistical analysis. B) Gmax/Gmax0 values for KCNQ1_T312C/KCNE1 and KCNQ1_I313S/KCNE1 (p=0.14 and p=0.10 respectively. (n= 3 oocytes). One-way ANOVA with Dunnett’s post-hoc multiple comparisons test was used for statistical analysis. C-D) Top view and side view of KCNQ1 channel with mutated residues highlighted.

Lin-Glycine does not increase the Po of KCNQ1_Y315F/KCNE1.

A-B) 10 consecutive traces of KCNQ1_Y315F/KCNE1 in A) control and B) in the presence of 20 μM Lin-Glycine. (top) Protocol used for the recordings. C-D) All-point amplitude histogram of 50 consecutive traces in C) control and D) Lin-Glycine). The single-channel current amplitude was reduced to 0.3 pA compared to 0.4 pA for WT KCNQ1/KCNE1 (cf. Figure 2C-D). Note that there were at least two channels in this patch. E) Average currents of 478 sweeps in control and 533 sweeps in Lin-Glycine. Note that panels A-D were all from the same patch.

Effect of Lin-Glycine in shifting the voltage-dependence of activation (ΔV0.5).

A similar effect in shifting the voltage-dependence of activation was found for KCNQ1_Y315F/KCNE1 and KCNQ1/KCNE1 after perfusion of several concentrations of Lin-Glycine. Comparisons at 20 μM of Lin-Glycine revealed no significant difference between the effect seen in KCNQ1/KCNE1 and KCNQ1_Y315F (p = 0.7435; n = 4 oocytes).

GV curves for KCNQ1 mutants.

GV curves in control (black) and after perfusion of 20 μM of Lin-Gly (red) for A) KCNQ1_D301E/KCNE1, B) KCNQ1_Y315F/KCNE1, C) KCNQ1_P320L/KCNE1, D) KCNQ1_T309S/KCNE1, E) KCNQ1_D317E/KCNE1, F) KCNQ1_I313S/KCNE1, and G) KCNQ1_T312S/KCNE1.

Conformational changes occurring at the pore during the transitions between non-conductive and conductive states.

A) The binding of PUFA to site II between K326 and D301 induces a series of interactions between residues near the external part of the selectivity filter. W304-D317 and W305-Y315 form a hydrogen bond (dash line, black). Furthermore, Y315 interacts also with T309 (dash line, black). P320 is reoriented to sit on top of W304 and W305 to favor a more stable configuration of the aromatic ring cuff. The result of those new interactions is a more stable and conductive pore. B) In the non-conductive state those interactions are likely to be absent and this results in a more unstable selectivity filter. Also, P320 is now flipped from its position on top of the two tryptophan of the aromatic ring cuff. C) Cryo-EM selectivity filter with S4 in the activated-state representative of a conductive selectivity filter. D) Cryo-EM selectivity filter with S4 in the resting state, representative of a non-conductive selectivity filter.

Comparison between the aromatic ring cuff configuration of Shaker and KCNQ1 channels.

A) In Shaker, P450 sits in between the two tryptophan and stabilizes the aromatic ring cuff. B) In contrast, in KCNQ1 the P320 is positioned further outward and away from the two tryptophan, generating a looser arrangement of the aromatic ring cuff.