Hiniesto-Iñigo I et al. (2025), Rescue of loss-of-function long QT syndrome-ass...

XB-ART-61299

Br J Pharmacol 2025 Mar 14; doi: 10.1111/bph.70008.

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Rescue of loss-of-function long QT syndrome-associated mutations in KV7.1/KCNE1 by the endocannabinoid N-arachidonoyl-L-serine (ARA-S).

Hiniesto-Iñigo I , Sridhar A , Louradour J , De la Cruz A , Lundholm S , Jauregi-Miguel A , Giannetti F , Sala L , Odening KE , Larsson HP , Ottosson NE , Liin SI .

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BACKGROUND AND PURPOSE: Congenital long QT syndrome (LQTS) involves genetic mutations affecting ion channels, leading to a prolonged QT interval and increased risk of potentially lethal ventricular arrhythmias. Mutations in the genes encoding KV7.1/KCNE1 are the most frequent, with channel loss-of-function contributing to LQTS. The endocannabinoid N-arachidonoyl-L-serine (ARA-S) has been shown to facilitate activation of wild type KV7.1/KCNE1 channels and to counteract a prolonged QT interval in isolated guinea pig hearts. In this study, we examine the ability of ARA-S to facilitate activation of LQTS-associated mutations, in various regions of the channel, and hence to counteract loss-of-function. EXPERIMENTAL APPROACH: The two-electrode voltage clamp technique on Xenopus oocytes expressing human KV7.1/KCNE1 channels was used to investigate the effects of ARA-S in 20 LQTS type 1-associated mutations distributed across the channel. Thereafter, different electrophysiology was used to assess ARA-S effects in mammalian cells. KEY RESULTS: ARA-S enhanced the function of all mutated channels by shifting V50 and increasing current amplitude. However, the magnitude of effect varied, related to whether mutations were in one of the two putative ARA-S binding sites on the channel as suggested by molecular dynamics simulations. ARA-S displayed translational potential by facilitating channel opening in mammalian cells and shortening the action potential duration in cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: This study demonstrates the rescuing capability of ARA-S on a diverse set of LQTS mutants. These insights may aid in developing drug compounds using ARA-S sites and mechanisms and guide interpretation of which LQTS mutants respond well to such compounds.

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Species referenced: Xenopus laevis
Genes referenced: kcne1

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	Graphical Abstract
	FIGURE 1ARA-S activates the WT KV7.1/KCNE1 channel using two putative binding sites. (a) Molecular structure of the endocannabinoid ARA-S. (b) Representative traces of WT KV7.1/KCNE1 currents under control conditions and in the presence of 3 and 10 μM ARA-S (grey, in control, and red traces, in 3 and 10 μM ARA-S, indicate an activating voltage step to +20 mV) and corresponding G(V) curve and normalised ITail for better visualisation of the V50 shift effect. For this specific cell: V50;ctrl = +25.3 mV, Itailmax;ctrl = 6.9 μA, V50; 3 μM ARA-S = +0.5 mV, Itailmax; 3 μM ARA-S = 9.1 μA, V50; 10 μM ARA-S = −29.3 mV, Itailmax; 10 μM ARA-S = 9.5 μA. Currents were generated in steps from −80 to +50 mV in 10 mV steps, followed by a tail voltage of −20 mV. The holding voltage was −80 mV. (c) Densities of ARA-S headgroup beads calculated from CG simulations of the KV7.1 structure (PDB: 6V00) illustrated at an isosurface value of 1.25 beads nm−3. (d) Normalised number of contacts between KV7.1 residues and ARA-S headgroup beads in CG simulations. A contact was assumed if a residue's bead was within 4.5 Å of an ARA-S bead and residues with a contact frequency >50% are coloured blue. (e) (left) Side view of the hKV7.1 (PDB: 6V00) indicating localisation of studied LQTS-associated mutations. One subunit of the channel is highlighted in grey. The cyan spheres highlight the location of the LQTS-associated mutations. (right) Side view of one subunit in which the residues have been coloured according to their location. Residues adjacent to the ARA-S Site I are coloured green, residues adjacent to the ARA-S Site II are coloured yellow, and other mutations are coloured blue.
	FIGURE 2ARA-S enhances the function of all LQTS mutants, with differences in the magnitude of effects. Effect of 3 and 10 μM ARA-S on KV7.1/KCNE1 LQTS mutants expressed in Xenopus oocytes and studied with the two-electrode voltage clamp technique. Representative examples of (a) Site I mutants KV7.1_I204F/KCNE1 and KV7.1_V212F/KCNE1, (b) Site II mutant KV7.1_R293H/KCNE1 and (c) ‘other mutant’ KV7.1_K196T/KCNE1. Currents under control conditions and in the presence of 3 and 10 μM ARA-S (grey, in control, and red traces, in 3 and 10 μM ARA-S, indicate an activating voltage step to +20 mV), corresponding G(V) curve and normalised ITail to the predicted top value for better visualisation of the V50 shift effect are shown. For the specific KV7.1_I204F/KCNE1 cell shown in Figure 2a, top: V50;ctrl = +36.0 mV, Itailmax;ctrl = 0.7 μA, V50; 3 μM ARA-S = +32.0 mV, Itailmax; 3 μM ARA-S = 1.4 μA, V50; 10 μM ARA-S = +20.3 mV, Itailmax; 10 μM ARA-S = 2.4 μA. Note that this is one of the cells with a less rightward shifted V50 to allow for clarity in visualising the Boltzmann fit. For the specific KV7.1_V212F/KCNE1 cell shown in Figure 2a, bottom: V50;ctrl = +50.7 mV, Itailmax;ctrl = 4.1 μA, V50; 3 μM ARA-S = +39.4 mV, Itailmax; 3 μM ARA-S = 5.7 μA, V50; 10 μM ARA-S = +23.6 mV, Itailmax; 10 μM ARA-S = 7.6 μA. Note that this representative cell exhibits a larger GMAX increase compared to the mean values. For the specific KV7.1_R293H/KCNE1 cell shown in Figure 2b: V50;ctrl = +28.2 mV, Itailmax;ctrl = 2.2 μA, V50; 3 μM ARA-S = +4.0 mV, Itailmax; 3 μM ARA-S = 5.1 μA, V50; 10 μM ARA-S = −15.4 mV, Itailmax; 10 μM ARA-S = 6.9 μA. For the specific KV7.1_K196T/KCNE1 cell shown in Figure 2c: V50;ctrl = + 57.0 mV, Itailmax;ctrl = 2.9 μA, V50; 3 μM ARA-S = +28.9 mV, Itailmax; 3 μM ARA-S = 5.6, V50; 10 μM ARA-S = 6.2 mV, Itailmax; 10 μM ARA-S = 7.7 μA. Currents were generated in steps from −80 to +70 mV in 10 mV steps, followed by a tail voltage of −20 mV. The holding voltage was −80 mV. For KV7.1_V212F/KCNE1, a pre-pulse to −120 mV was used to force the closing of the channel before the activation steps. (d) Mean ΔV50, (e) ΔGMAX and (f) ΔΔG0 induced by 3 μM (left) and 10 μM (right) of ARA-S in indicated LQTS mutants. Black bar represents WT, green bars indicate mutants located near binding Site I, yellow bars indicate mutants located near binding Site II, and blue bars indicate mutations located outside putative binding sites. Dashed line denotes the effect of ARA-S in the WT KV7.1/KCNE1 channel (from Hiniesto-Iñigo et al., 2023). Because of the abnormal biophysical behaviour of S330F, with a prominent leak component, ΔGMAX was calculated as the ARA-S effect on ΔGMAX − ΔGMIN. This alternative analysis, which quantifies the voltage-dependent increase in G, is denoted as S330F*. ΔΔG0 was calculated as described in Section 2. Statistics indicate one-way ANOVA with Bonferroni's multiple comparison test and indicates the difference compared to the effect of ARA-S in WT. Only significant differences are indicated. All other comparisons are non-significant (ns). Data are shown as mean ± SEM; n = 5–21.
	FIGURE 3Impairment of ARA-S binding by the aromatic rings in I204F and V212F. (a) Preferential residence sites of the ARA-S headgroups and tail from atomistic simulations illustrated in red and purple, respectively. The I204 and V212 residues are shown as green spheres. (b) Illustration of the hydrophobic pocket adjacent to the I204 and V212 residues permitting the preferential localisation of the ARA-S tail. (c) Stability of ARA-S at the VSD–PD interface within atomistic simulations of the WT, I204F and V212F KV7.1 channels assessed as the fraction of frames belonging to each cluster. For each simulation set, the most populated ARA-S cluster is illustrated in the inset. (d) Relative contact frequencies of ARA-S atoms with VSD (left) and PD (right) residues compared between simulations of the WT, I204F and V212F KV7.1 channels. A value greater than 1 signifies a weakening of interaction in comparison to WT (i.e. ARA-S atoms have weaker interaction with the mutant than WT) and vice versa. Cyan and orange circles illustrate the reduced interactions of the ARA-S tail and head groups, respectively.
	FIGURE 4Different concentrations of ARA-S are needed to rescue the LQTS mutants. Rescuing effect of ARA-S on LQTS mutants expressed in Xenopus oocytes and studied with the two-electrode voltage clamp technique, by combining the properties of the LQTS mutant and pharmacological ARA-S effect. (a) Mean V50 of indicated mutants. Dashed line denotes the V50 of the WT KV7.1/KCNE1 channel (+30 mV). Statistics indicate one-way ANOVA with Bonferroni's multiple comparison test and indicates the significance of the difference compared to the WT. Data are shown as mean ± SEM; n = 5–152. (b) Mean V50 of the LQTS mutants under control conditions (black bar, similar as in Figure 4a) and after the application of 3 μM ARA-S (pink bar) and 10 μM ARA-S (red bar). The dashed line denotes the V50 of WT KV7.1/KCNE1 under control conditions (without ARA-S; +30 mV). (c) Mean Iamp at +20 mV of indicated mutants. Dashed line denotes 100% of current amplitude for the WT in time-matched experiments (see Supporting Information Methods). Statistics indicate one-way ANOVA with Bonferroni's multiple comparison test and indicates the significance of the difference compared to the WT. Data are shown as mean ± SEM; n = 6–43. (d) Relative Iamp, 20 mV of LQTS-associated mutants co-expressed with KCNE1 under control conditions (black, similar as in Figure 4c), in the presence of 3 μM ARA-S (pink bar) and 10 μM ARA-S (red bar). 100% denotes the current for WT KV7.1/KCNE1 under control conditions (without ARA-S). The mean ARA-S induced increase in current amplitude for each mutant is multiplied with the control amplitude for each mutant. Data are shown as mean ± SEM; n = 5–21. Only significant differences are indicated. All other comparisons are non-significant (ns). Please note that Figure 4b and 4d does not include statistical analysis. This is because the rescuing capability of ARA-S in these panels were assessed by mathematically estimating the combined burden of a mutant and ARA-S. As a result, no direct statistical comparisons were made in these instances.
	FIGURE 5ARA-S facilitates activation of WT and mutant KV7.1/KCNE1 in mammalian cells and shortens LQT1 rabbit ventricular AP duration. (a) Cartoon of automated patch-clamp and representative traces of WT KV7.1/KCNE1 currents from CHO cells under control conditions and in the presence of 10 μM ARA-S (grey, in control, and red trace, in ARA-S, indicate an activating voltage step to −20 mV) and corresponding G(V) curve. For this specific cell: V50;ctrl = −4.7 mV, Itailmax;ctrl = 7.8 nA, V5duration0; 10 μM ARA-S = −29.3 mV, Itailmax; 10 μM ARA-S = 9.6 nA. Currents were generated in steps from −100 to +60 mV in 20 mV steps, followed by a tail voltage of −20 mV. The holding voltage was −90 mV. (b) Concentration–response curve showing the ΔV50 effect of ARA-S on WT KV7.1/KCNE1 in CHO cells. The curves represent the best fit of Equation (4) in Supporting Information Methods. Best fit for 22°C: EC50 = 15.0 μM, ΔV50, max = −72.8 mV. Best fit for 35°C: EC50 = 26.4 μM, ΔV50, max = −78.6 mV. Statistics and n-values are indicated in Table S6. Data are shown as mean ± SEM. Small error bars are covered by symbols. (c) Cartoon of manual patch-clamp in HEK293 cells and representative traces of WT KV7.1/KCNE1 currents from HEK293 cells under control conditions and in the presence of 3 μM ARA-S (grey, in control, and red trace, in ARA-S, indicate an activating voltage step to +20 mV) and corresponding G(V) curve. For this specific cell: V50;ctrl = +55.0 mV, Itailmax;ctrl = 0.4 nA, V50; 3 μM ARA-S = +43.8 mV, Itailmax; 3 μM ARA-S = 0.4 nA. Currents were generated in steps from −80 to +100 mV in 20 mV steps, followed by a tail voltage of −40 mV. The holding voltage was −90 mV. (d) Same as in Figure 5c but for KV7.1(G269S)/KV7.1/KCNE1. For this specific cell: V50;ctrl = +46.0 mV, Itailmax;ctrl = 0.2 nA, V50; 3 μM ARA-S = +37.7 mV, Itailmax; 3 μM ARA-S = 0.2 nA. (e) Mean ΔV50 and (f) ΔIamp, 20 mV induced by 3 μM of ARA-S in WT and KV7.1(G269S)/KV7.1/KCNE1. Data are shown as mean ± SEM; n = 5–6. Statistics indicate one sample t-test to compare to a hypothetical value of 0. A t-test comparison between WT and the G269S mutant showed no statistical difference between the groups (ns). Only significant differences are indicated. All other comparisons are non-significant (ns). (g) Cartoon of manual patch-clamp in rabbit cardiomyocytes and representative action potential trace before (ctrl, black line) and after perfusion of 10 μM ARA-S (red line). (h) Corresponding time course of the APD90 over time obtained in an exemplary left ventricular cardiomyocyte from LQT1 rabbit heart. (i) Average APD90 between control condition and 10 μM ARA-S. Data are shown as mean ± SEM; n = 7 cells, n = 2 rabbits. Statistics indicate non-parametric paired Wilcoxon test.
	Supplementary Figure 1. Coarse-grain (CG) parameters for ARA-S. A) Illustration of the coarse-grain Martini bead mapping of ARA-S. The beads were mapped onto the centre-of-geometry of their constituent heavy-atoms and associated hydrogens. B) Top: For the validation of inter-bead bond parameters, the distribution of distances between the centre-of-geometries of the beads' constituent atoms were calculated from atomistic simulations (green) and compared against distances within CG simulations (red). Bottom: For the validation of inter-bead angle parameters, the distribution of angles between the centre-of-geometries of beads' constituent atoms were calculated from atomistic simulations (green) and compared against angles within CG simulations (red). C) For the validation of bead-types, the positioning of the head-group beads relative to the centre of the bilayer was compared between atomistic (green) and CG (red) simulations. For comparison, the positioning of the POPC phosphocholine is illustrated as a shaded grey area.
	Supplementary Figure 2. Representative traces of KV7.1/KCNE1 LQTS mutations. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. Representative traces of indicated currents under control conditions and in the presence of 3 and 10 µM ARA-S (grey, in control, and red traces, in 3 and 10 µM ARA-S, indicate an activating voltage step to +20 mV) and corresponding G(V) curves. For these specific cells: R190Q: V50;ctrl = +41.6 mV; Itailmax;ctrl = 2.7 µA , V50; 3 µM ARA-S = +14.1 mV, Itailmax; 3 µM ARA-S = 3.7 µA, V50; 10 µM ARA-S = - 12.2 mV, Itailmax; 10 µM ARA-S = 5.0 µA. V215M: V50;ctrl = +39.4 mV, Itailmax;ctrl = 6.3 µA, V50; 3 µM ARA-S = +33.3 mV, Itailmax; 3 µM ARA-S =7.9 µA, V50; 10 µM ARA-S = -3.4 mV, Itailmax; 10 µM ARA-S = 9.4 µA. S225L: V50;ctrl = +41.4 mV, Itailmax;ctrl = 0.7 µA, V50; 3 µM ARA-S = +25.7 mV, Itailmax; 3 µM ARA-S = 2.4 µA, V50; 10 µM ARA-S = +1.4 mV, Itailmax; 10 µM ARA-S = 3.1 µA. G269S: V50;ctrl = +20.9 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +12.0 mV, Itailmax; 3 µM ARA-S = 0.7 µA, V50; 10 µM ARA-S = +0.1 mV, Itailmax; 10 µM ARA-S = 0.9 µA. Y278H: V50;ctrl = +28.9 mV, Itailmax;ctrl = 0.5 µA , V50; 3 µM ARA-S = +15.1 mV, Itailmax; 3 µM ARA-S = 0.6 µA, V50; 10 µM ARA-S = -8.5 mV, Itailmax; 10 µM ARA-S = 0.8 µA. Y281C: V50;ctrl = +37.0 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +12.6 mV, Itailmax; 3 µM ARA-S = 1.0 µA, V50; 10 µM ARA-S =- 14.4 mV, Itailmax; 10 µM ARA-S = 1.4 µA. L282F: V50;ctrl = +34.9 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +20.7 mV, Itailmax; 3 µM ARA-S = 0.6 µA, V50; 10 µM ARA-S = -7.2 µA, Itailmax; 10 µM ARA-S = 0.7 µA. L282P: V50;ctrl = +36.2 mV, Itailmax;ctrl = 0.4 µA, V50; 3 µM ARA-S = +24.7 mV, Itailmax; 3 µM ARA-S = 0.5 µA, V50; 10 µM ARA-S = -2.3 mV, Itailmax; 10 µM ARA-S = 0.6 µA. A287E: V50;ctrl = +20.1 mV, Itailmax;ctrl =0.5 µA, V50; 3 µM ARA-S = +8.2 mV, Itailmax; 3 µM ARA-S = 0.7 µA, V50; 10 µM ARA-S = -5.8 mV, Itailmax; 10 µM ARA-S = 0.8 µA. R293C: V50;ctrl = +39 mV, Itailmax;ctrl = 6.5 µA, V50; 3 µM ARA-S = +14.5 mV, Itailmax; 3 µM ARA-S = 9.5 µA, V50; 10 µM ARA-S = -12.6 mV, Itailmax; 10 µM ARA-S = 10.5 µA. A300T: V50;ctrl = +51.2 mV, Itailmax;ctrl = 0.9 µA, V50; 3 µM ARA-S = +14.0 mV, Itailmax; 3 µM ARA-S = +2.1 µA, V50; 10 µM ARA-S = - 17.5 mV, Itailmax; 10 µM ARA-S = 2.4 µA. D301V: V50;ctrl = +20.3 mV, Itailmax;ctrl = 0.3 µA, V50; 3 µM ARA-S = +2.9 mV, Itailmax; 3 µM ARA-S = 0.4 µA, V50; 10 µM ARA-S = -12.9 mV, Itailmax; 10 µM ARA-S = 0.4 µA. S330F: V50;ctrl = +26.9 mV, Itailmax;ctrl = 0.3 µA, V50; 3 µM ARA-S = +14.8 mV, Itailmax; 3 µM ARA-S = 0.4 µA, V50; 10 µM ARA-S = 0 mV, Itailmax; 10 µM ARA-S = 0.5 µA. R366Q: V50;ctrl = +24.3 mV, Itailmax;ctrl = 0.7 µA, V50; 3 µM ARA-S = +2.1 mV, Itailmax; 3 µM ARA-S = 1.6 µA, V50; 10 µM ARA-S = -12.6 mV, Itailmax; 10 µM ARA-S = 2.2 µA. A370V: V50;ctrl = +41.0 mV, Itailmax;ctrl = 7.0 µA, V50; 3 µM ARA-S = +15.3 mV, Itailmax; 3 µM ARA-S = 9.8 µA, V50; 10 µM ARA-S = -20.7 mV, Itailmax; 10 µM ARA-S = 12.1 µA. R555C: V50;ctrl = +30.2 mV, Itailmax;ctrl = 7.4 µA, V50; 3 µM ARA-S = -4.8 mV, Itailmax; 3 µM ARA-S = 11.0 µA, V50; 10 µM ARA-S = - 18.2 mV, Itailmax; 10 µM ARA-S = 11.2 µA. Currents were generally generated in steps from -80 to +50 or +70 mV in 10 mV steps, followed by a tail voltage of -20 mV. Values on the top part of the G(V) curve that decayed have been excluded from the representative traces and the G(V) curve.
	Supplementary Figure 2. Representative traces of KV7.1/KCNE1 LQTS mutations. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. Representative traces of indicated currents under control conditions and in the presence of 3 and 10 µM ARA-S (grey, in control, and red traces, in 3 and 10 µM ARA-S, indicate an activating voltage step to +20 mV) and corresponding G(V) curves. For these specific cells: R190Q: V50;ctrl = +41.6 mV; Itailmax;ctrl = 2.7 µA , V50; 3 µM ARA-S = +14.1 mV, Itailmax; 3 µM ARA-S = 3.7 µA, V50; 10 µM ARA-S = - 12.2 mV, Itailmax; 10 µM ARA-S = 5.0 µA. V215M: V50;ctrl = +39.4 mV, Itailmax;ctrl = 6.3 µA, V50; 3 µM ARA-S = +33.3 mV, Itailmax; 3 µM ARA-S =7.9 µA, V50; 10 µM ARA-S = -3.4 mV, Itailmax; 10 µM ARA-S = 9.4 µA. S225L: V50;ctrl = +41.4 mV, Itailmax;ctrl = 0.7 µA, V50; 3 µM ARA-S = +25.7 mV, Itailmax; 3 µM ARA-S = 2.4 µA, V50; 10 µM ARA-S = +1.4 mV, Itailmax; 10 µM ARA-S = 3.1 µA. G269S: V50;ctrl = +20.9 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +12.0 mV, Itailmax; 3 µM ARA-S = 0.7 µA, V50; 10 µM ARA-S = +0.1 mV, Itailmax; 10 µM ARA-S = 0.9 µA. Y278H: V50;ctrl = +28.9 mV, Itailmax;ctrl = 0.5 µA , V50; 3 µM ARA-S = +15.1 mV, Itailmax; 3 µM ARA-S = 0.6 µA, V50; 10 µM ARA-S = -8.5 mV, Itailmax; 10 µM ARA-S = 0.8 µA. Y281C: V50;ctrl = +37.0 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +12.6 mV, Itailmax; 3 µM ARA-S = 1.0 µA, V50; 10 µM ARA-S =- 14.4 mV, Itailmax; 10 µM ARA-S = 1.4 µA. L282F: V50;ctrl = +34.9 mV, Itailmax;ctrl = 0.5 µA, V50; 3 µM ARA-S = +20.7 mV, Itailmax; 3 µM ARA-S = 0.6 µA, V50; 10 µM ARA-S = -7.2 µA, Itailmax; 10 µM ARA-S = 0.7 µA. L282P: V50;ctrl = +36.2 mV, Itailmax;ctrl = 0.4 µA, V50; 3 µM ARA-S = +24.7 mV, Itailmax; 3 µM ARA-S = 0.5 µA, V50; 10 µM ARA-S = -2.3 mV, Itailmax; 10 µM ARA-S = 0.6 µA. A287E: V50;ctrl = +20.1 mV, Itailmax;ctrl =0.5 µA, V50; 3 µM ARA-S = +8.2 mV, Itailmax; 3 µM ARA-S = 0.7 µA, V50; 10 µM ARA-S = -5.8 mV, Itailmax; 10 µM ARA-S = 0.8 µA. R293C: V50;ctrl = +39 mV, Itailmax;ctrl = 6.5 µA, V50; 3 µM ARA-S = +14.5 mV, Itailmax; 3 µM ARA-S = 9.5 µA, V50; 10 µM ARA-S = -12.6 mV, Itailmax; 10 µM ARA-S = 10.5 µA. A300T: V50;ctrl = +51.2 mV, Itailmax;ctrl = 0.9 µA, V50; 3 µM ARA-S = +14.0 mV, Itailmax; 3 µM ARA-S = +2.1 µA, V50; 10 µM ARA-S = - 17.5 mV, Itailmax; 10 µM ARA-S = 2.4 µA. D301V: V50;ctrl = +20.3 mV, Itailmax;ctrl = 0.3 µA, V50; 3 µM ARA-S = +2.9 mV, Itailmax; 3 µM ARA-S = 0.4 µA, V50; 10 µM ARA-S = -12.9 mV, Itailmax; 10 µM ARA-S = 0.4 µA. S330F: V50;ctrl = +26.9 mV, Itailmax;ctrl = 0.3 µA, V50; 3 µM ARA-S = +14.8 mV, Itailmax; 3 µM ARA-S = 0.4 µA, V50; 10 µM ARA-S = 0 mV, Itailmax; 10 µM ARA-S = 0.5 µA. R366Q: V50;ctrl = +24.3 mV, Itailmax;ctrl = 0.7 µA, V50; 3 µM ARA-S = +2.1 mV, Itailmax; 3 µM ARA-S = 1.6 µA, V50; 10 µM ARA-S = -12.6 mV, Itailmax; 10 µM ARA-S = 2.2 µA. A370V: V50;ctrl = +41.0 mV, Itailmax;ctrl = 7.0 µA, V50; 3 µM ARA-S = +15.3 mV, Itailmax; 3 µM ARA-S = 9.8 µA, V50; 10 µM ARA-S = -20.7 mV, Itailmax; 10 µM ARA-S = 12.1 µA. R555C: V50;ctrl = +30.2 mV, Itailmax;ctrl = 7.4 µA, V50; 3 µM ARA-S = -4.8 mV, Itailmax; 3 µM ARA-S = 11.0 µA, V50; 10 µM ARA-S = - 18.2 mV, Itailmax; 10 µM ARA-S = 11.2 µA. Currents were generally generated in steps from -80 to +50 or +70 mV in 10 mV steps, followed by a tail voltage of -20 mV. Values on the top part of the G(V) curve that decayed have been excluded from the representative traces and the G(V) curve.
	Supplementary Figure 3. Effects on ΔIamp at +20 mV by 3 and 10 µM ARA-S on KV7.1/KCNE1 LQTS mutations. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. Mean ΔIamp at +20 mV induced by 3 µM (left) and 10 µM (right) of ARA-S in LQTS mutations. Black bar represents WT, green bars indicate mutants located in binding Site I, yellow bars indicate mutants located in binding Site II and light blue bars represent mutations located outside the binding sites. The dashed line denotes the effect of ARA-S on Iamp at +20 mV in the WT KV7.1/KCNE1 channel. Statistics indicate one-way ANOVA with Bonferroni´s multiple comparison test and indicate the difference compared to the effect of ARA-S in WT KV7.1/KCNE1. Only significant differences are indicated. All other comparisons are non-significant (ns). Data shown as mean ± SEM; n = 5-21.
	Supplementary Figure 4: Relation between intrinsic V50 and the ARA-S V50 effect in mutants I204F and V212F. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. V50 induced by 3 µM (left) or 10 µM (right) of ARA-S as a function of the intrinsic V50 of the cell for A) I204F and B) V212F. The lines denote simple linear regression. Best fit for KV7.1_I204F/KCNE1 and for 3 µM: slope = 0.029; R2 = 0.002. Best fit for KV7.1_I204F/KCNE1 and for 10 µM: slope = -0.46; R2 = 0.21. Best fit for KV7.1_V212F/KCNE1 and for 3 µM: slope = -0.009; R2 for 3 µM = <0.0002. Best fit for KV7.1_V212F/KCNE1 and for 10 µM: slope = - 0.31; R2 = 0.12. Although there is biological variability in the data, there is no clear correlation between the ARA-S response and the intrinsic V50, which supports that the reduced V50 response of I204F and V212F is not caused by analysis uncertainties. If anything, there is a tendency to larger estimated V50 shifts with a more depolarized V50 for 10 µM ARA-S.
	Supplementary Figure 5: Mean V50 of indicated mutants for batch and time-matched experiments. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. The dashed line denotes the V50 of the WT KV7.1/KCNE1 channel (+30 mV). Statistics indicate one-way ANOVA with Bonferroni´s multiple comparison test and indicates the significance of the difference compared to the WT. Data shown as mean ± SEM; n = 5-6.
	Supplementary Figure 6. Concentration-response curves of how V50 of mutants K196T and I204F change in the presence of ARA-S. Data from human KV7.1/KCNE1 channels expressed in Xenopus oocytes and channels studied using two-electrode voltage clamp. A-B) Concentration-response curve showing how V50 of the LQTS mutant (A) K196T and (B) I204F shift with increasing concentrations of ARA-S. The curves start at the intrinsic V50 of each mutant (i.e. without ARA-S). The data shows how the V50 becomes gradually less depolarized with increasing concentrations of ARA-S. Data shown as mean ± SEM; n = 6-17. Small error bars are covered by symbols. The curves represent the best fit of equation 4 in SI Materials and Methods. Best fit for KV7.1_K196T/KCNE1: EffectMAX = -8 mV; EC50 = 3.7 µM. Best fit for KV7.1_I204F/KCNE1: EffectMAX = -30 mV; EC50 = 12.6 µM. The dashed line represents the average V50 of the WT KV7.1/KCNE1 channel (+30 mV).
	Supplementary Figure 7. Effect of heterozygous conditions on KV7.1/KCNE1 mutants’ intrinsic behaviour and response to ARA-S. Heterozygous conditions were mimicked by co-injecting WT and mutant RNA, as described in Material and Methods, in Xenopus oocytes and channels studied usingtwo-electrode voltage clamp. Homozygous conditions are represented by uniform bars and heterozygous conditions represented by dashed bars. A) Comparison of the mean V50. Dashed line denotes the V50 of the WT KV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-152. B) Mean Iamp at +20 mV. Dashed line denotes 100% of the current in WT KV7.1/KCNE1. Heterozygous vs WT expression was time-matched. Data shown as mean ± SEM; n = 5-19. C) Comparison of mean ΔV50 or ΔΔG0 induced by 3 µM (left) and 10 µM (right) of ARA-S. ΔΔG0 was calculated as described in Materials and Methods. Black bar represents WT, green bars indicate mutants located in binding Site I, yellow bars indicate mutants located in binding Site II and blue bar represents mutations located outside putative binding sites. Dashed line denotes the effect of ARA-S in the WT KV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-16. D) Mean V50 of the LQTS mutants under control conditions (black bar) and after the application of 3 µM ARA-S (pink bar) and 10 µM ARA-S (red bar). The dashed line denotes the V50 of WT KV7.1/KCNE1 under control conditions (without ARA-S; +30 mV). Data shown as mean ± SEM; n = 5-152. E) Comparison of the mean ΔIamp at +20 mV induced by 3 µM (left) and 10 µM (right) of ARA-S in indicated LQTS mutants during homozygous conditions (uniform bar) and heterozygous conditions (dashed bar). Same colour coding as in C. The dashed line denotes the effect of ARA-S on Iamp, +20 mV in the WT hKV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-19. F) Relative Iamp, 20 mV of LQTS-associated mutants co-expressed with KCNE1 under control conditions (black), in the presence of 3 µM ARA-S (pink bar) and 10 µM ARA-S (red bar). 100% denotes the current for WT KV7.1/KCNE1 under control conditions (without ARA-S). The mean ARA-S induced increase in current amplitude for each mutant is multiplied with the control amplitude for each mutant. Dashed line denotes the effect of ARA-S in the WT KV7.1/KCNE1 channel (Hiniesto-Iñigo et al., 2023). Data shown as mean ± SEM; n = 5-19. Statistics in A-B indicate one-way ANOVA with Bonferroni´s multiple comparison test and indicate differences compared to the properties of WT KV7.1/KCNE1 or the properties between homozygous and heterozygous conditions (indicated by a line connecting two bars). Statistics in panels C and E indicate one-way ANOVA with Bonferroni´s multiple comparison tests and indicate the difference compared to the value of the effect of ARA-S in KV7.1/KCNE1 or the effect of ARA-S between homozygous and heterozygous conditions (indicated by a line connecting two bars). Only significant differences are indicated. All other comparisons are non-significant (ns). Please note that panels D and F do not include statistical analysis. This is because the rescuing capability of ARA-S in these panels were assessed by mathematically estimating the combined burden of a mutant and ARA-S. As a result, no direct statistical comparisons were made in these instances.
	Supplementary Figure 7. Effect of heterozygous conditions on KV7.1/KCNE1 mutants’ intrinsic behaviour and response to ARA-S. Heterozygous conditions were mimicked by co-injecting WT and mutant RNA, as described in Material and Methods, in Xenopus oocytes and channels studied usingtwo-electrode voltage clamp. Homozygous conditions are represented by uniform bars and heterozygous conditions represented by dashed bars. A) Comparison of the mean V50. Dashed line denotes the V50 of the WT KV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-152. B) Mean Iamp at +20 mV. Dashed line denotes 100% of the current in WT KV7.1/KCNE1. Heterozygous vs WT expression was time-matched. Data shown as mean ± SEM; n = 5-19. C) Comparison of mean ΔV50 or ΔΔG0 induced by 3 µM (left) and 10 µM (right) of ARA-S. ΔΔG0 was calculated as described in Materials and Methods. Black bar represents WT, green bars indicate mutants located in binding Site I, yellow bars indicate mutants located in binding Site II and blue bar represents mutations located outside putative binding sites. Dashed line denotes the effect of ARA-S in the WT KV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-16. D) Mean V50 of the LQTS mutants under control conditions (black bar) and after the application of 3 µM ARA-S (pink bar) and 10 µM ARA-S (red bar). The dashed line denotes the V50 of WT KV7.1/KCNE1 under control conditions (without ARA-S; +30 mV). Data shown as mean ± SEM; n = 5-152. E) Comparison of the mean ΔIamp at +20 mV induced by 3 µM (left) and 10 µM (right) of ARA-S in indicated LQTS mutants during homozygous conditions (uniform bar) and heterozygous conditions (dashed bar). Same colour coding as in C. The dashed line denotes the effect of ARA-S on Iamp, +20 mV in the WT hKV7.1/KCNE1 channel. Data shown as mean ± SEM; n = 5-19. F) Relative Iamp, 20 mV of LQTS-associated mutants co-expressed with KCNE1 under control conditions (black), in the presence of 3 µM ARA-S (pink bar) and 10 µM ARA-S (red bar). 100% denotes the current for WT KV7.1/KCNE1 under control conditions (without ARA-S). The mean ARA-S induced increase in current amplitude for each mutant is multiplied with the control amplitude for each mutant. Dashed line denotes the effect of ARA-S in the WT KV7.1/KCNE1 channel (Hiniesto-Iñigo et al., 2023). Data shown as mean ± SEM; n = 5-19. Statistics in A-B indicate one-way ANOVA with Bonferroni´s multiple comparison test and indicate differences compared to the properties of WT KV7.1/KCNE1 or the properties between homozygous and heterozygous conditions (indicated by a line connecting two bars). Statistics in panels C and E indicate one-way ANOVA with Bonferroni´s multiple comparison tests and indicate the difference compared to the value of the effect of ARA-S in KV7.1/KCNE1 or the effect of ARA-S between homozygous and heterozygous conditions (indicated by a line connecting two bars). Only significant differences are indicated. All other comparisons are non-significant (ns). Please note that panels D and F do not include statistical analysis. This is because the rescuing capability of ARA-S in these panels were assessed by mathematically estimating the combined burden of a mutant and ARA-S. As a result, no direct statistical comparisons were made in these instances.
	Supplementary Figure 8. Effect of ARA-S on human WT KV7.1/KCNE1 expressed in CHO cells using automated patch-clamp. A) Representative traces of the re-application of ARA-S under control conditions and in the presence of 10 µM ARA-S at 22°C (grey, in control, and red traces, in 10 µM ARA-S, indicate an activating voltage step to -20 mV) and corresponding G(V) curves. B) ΔV50 plotted against the re-application of the indicated concentration of ARA-S at 22°C. 4-5 repeated applications for about 5-7 minutes of ARA-S were necessary until a maximized, stable response was obtained. n ranges from 1 to 14, depending on the survival of cells. See Supplementary Table VI for n values used for quantification. C) Representative traces of ARA-S under control conditions and in the presence of 10 µM ARA-S at 35°C (grey, in control, and red traces in 10 µM ARA-S indicate an activating voltage step to -20 mV) and corresponding G(V) curve. For this specific cell: V50;ctrl = -22.9 mV, Itailmax;ctrl = 4.3 nA, V50; 10 µM ARA-S = -40.0 mV, Itailmax; 10 µM ARA-S = 3.9 nA, V50; 10 µM ARA-S;normalized = -40.0 mV, Itailmax; 10 µM ARA-S;normalized = 7.3 nA. D) Concentration-response curve of the ΔGMAX effects of ARA-S in 22°C and 35°C in CHO cells. E) Concentration-response curve of the comparison of the ΔV50 ARA-S effects between oocytes in TEVC (from (Hiniesto-Iñigo et al., 2023)), CHO cells at 22°C and 35°C, and WT and G269S mutant in HEK293 cells. Data shown as mean ± SEM; n is found in Main Figure 5 and Supplementary Table VI.
	Supplementary Figure 9. Effect of ARA-S on LQT1 rabbit cardiomyocytes. (A-C) Average values between control condition and 10 µM ARA-S for A) resting membrane potential RMP, B) maximal velocity of the upstroke and C) amplitude of the AP. Statistics indicate paired t-test. ns = nonsignificant (n = 7 cells, N = 2 rabbits). D) Correlation between the ARA-S-induced decrease in APD90 and the APD90 at baseline, analysed with a Spearman correlation test.