BACKGROUND AND PURPOSE:Mutations in both KCNQ1 and KCNE1, which together form the cardiac IKs current, are associated with inherited conditions such as long and short QT syndromes. Mefenamic acid, a non-steroidal anti-inflammatory drug, is an IKs potentiator and may be utilised as an archetype to design therapeutically useful IKs agonists. However, here we show that mefenamic acid can also act as an IKs inhibitor, and our data reveal its dual effects on KCNQ1/KCNE1 channels. EXPERIMENTAL APPROACH:Effects of mefenamic acid on wild type (WT) and mutant KCNQ1/KCNE1 channels expressed in tsA201 cells were studied using whole cell patch clamp. Molecular dynamics simulations were used to determine trajectory clustering. KEY RESULTS:Mefenamic acid inhibits WT IKs at high concentrations while preserving some attributes of current potentiation. Inhibitory actions of mefenamic acid are unmasked at lower drug concentrations by KCNE1 and KCNQ1 mutations in the mefenamic acid binding pocket, at the extracellular end of KCNE1 and in the KCNQ1 S6 helix. Mefenamic acid does not inhibit KCNQ1 in the absence of KCNE1 but inhibits IKs current in a concentration-dependent manner in the mutant channels. Inhibition involves modulation of pore kinetics and/or voltage sensor domain-pore coupling in WT and in the KCNE1 E43C mutant. CONCLUSION AND IMPLICATIONS:This work highlights the importance of structural motifs at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channels, and their interactions, in determining the nature of drug effects on the IKs channel complex and has important implications for treating patients with specific long QT mutations.

The IKs current formed by the co-assembly of KCNE1 and KCNQ1 plays an important role in cardiac repolarization. Mefenamic acid, an NSAID, is known to enhance IKs currents and has in turn been suggested as a therapeutic starting point for the development of compounds for the treatment of long QT syndrome. Our previous examinations of mefenamic acid’s action revealed that residue K41 on KCNE1 was critical for mefenamic acid’s activating effect on fully KCNE1 saturated, and partially saturated IKs channel complexes. The present study extends our previous work by incorporating the K41C-KCNE1 mutation into individual subunits to destabilize local mefenamic acid binding and explore how many of the remaining mefenamic acid-bound WT KCNE1-KCNQ1 subunits are required to support the activating action of the drug. Our results show that the potency of mefenamic acid action is reduced by the presence of K41C-KCNE1 subunits in a graded and stoichiometric, but non-linear manner. Modeling results are consistent with the idea that WT IKs subunits, in the presence of mefenamic acid, precede activation of K41C-IKs subunits due to their augmented voltage sensor kinetics.

In cardiomyocytes, Kv7.1 associates with the regulatory subunit KCNE1 to generate the delayed rectifier potassium current IKs, which plays a crucial role in cardiac repolarization at elevated heart rates. Gain-of-function mutations in either of these subunits are associated with short QT syndrome, a condition that increases the risk of cardiac arrhythmias including atrial fibrillation, syncope, and sudden death. Therefore, the study of pharmacological inhibitors of Kv7.1 and IKs is of significant therapeutic interest. In this work, we used whole-cell patch clamp recordings to characterize the electrophysiological effects of the Kv7.1 blocker 3-(triphenylmethylaminomethyl)pyridine (UCL2077) in both Kv7.1 and IKs (Kv7.1 + KCNE1) channels. We found that UCL2077 inhibited both Kv7.1 and IKs channels with high affinity (IC50 in the picomolar range) and mild voltage dependence. The drug induced a biphasic time-dependent current decay and reduced current reactivation of Kv7.1, whereas the kinetics of IKs were unaffected. We examined state dependence using mutations that functionally stabilize Kv7.1/IKs in either the intermediate-open (E160R/R231E) or in the activated-open (E160R/R237E) state. In both channels, UCL2077 potency correlated with the strength of the electromechanical coupling. Our results are further supported by a kinetic Markov model simulating UCL2077 binding that closely resembles the experimental currents. Overall, our work provides an in-depth characterization of UCL2077's action on Kv7.1 and IKs channels, offering valuable insights for the development of Kv7.1/IKs inhibitors in the context of short QT syndrome and other cardiac arrhythmias. SIGNIFICANCE STATEMENT: This study characterizes UCL2077: a highly specific, high-affinity inhibitor of Kv7.1 and IKs channels with therapeutic implications for cardiac arrhythmia. Our work reveals a picomolar affinity, mild voltage dependence, and a kinetic modulation consistent with state-dependent open-channel block.

Voltage-gated potassium ion (K+) channels perform critical roles in many physiological processes, while gain- or loss-of-function mutations lead to life-threatening pathologies. Here, we establish the high-resolution structure of a pivotal intermediate state of the Kv7.1 (KCNQ1) channel using cryogenic electron microscopy. The 3.53 Å resolution structure reveals straightened upper S1 and S2 voltage sensor helices, distancing them from the pore filter helix compared to fully activated channels. The outward translation of the S4 voltage sensor is essentially complete in this intermediate state, and the S4-S6 helices and the S4-S5 linker do not change position significantly between intermediate and activated states. The PIP2 ligand can bind in both states. Movement of S1 and S2 helices towards the filter helix from intermediate to activated states may explain smaller components of KCNQ1 voltage sensor fluorescence, differential Rb+/K+ selectivity, and pharmacological responses to activators and inhibitors. Single channel recordings and the location of long QT mutations suggest the potential physiological and disease importance of the intermediate state.

KCNQ2 channel subunits form part of the M-current and underlie one of the major potassium currents throughout the human nervous system, regulating resting membrane potentials, shaping action potentials, and impeding repetitive neuronal firing. However, how individual subunits within tetramers control channel functionality remains unresolved. Here, we investigate (i) whether opening of KCNQ2 channels requires a concerted step or can result from independent subunit activation and (ii) how individual subunits regulate gate opening and conductance. The E140R mutation in the S2 segment prevents activated voltage sensor conformations, but concatemeric constructs containing up to three E140R subunits retain KCNQ2-like currents. The underlying single-channel currents show subconductance levels resulting from limitations in inner gate dimensions, determined by the number of activated subunits and their spatial arrangement. Channel opening is allosteric and requires activation of only a single subunit, which can accentuate the influence of clinically relevant heterozygous mutations at threshold voltages.

The ion-conducting IKs channel complex, important in cardiac repolarization and arrhythmias, comprises tetramers of KCNQ1 α-subunits along with 1–4 KCNE1 accessory subunits and calmodulin regulatory molecules. The E160R mutation in individual KCNQ1 subunits was used to prevent activation of voltage sensors and allow direct determination of transition rate data from complexes opening with a fixed number of 1, 2, or 4 activatable voltage sensors. Markov models were used to test the suitability of sequential versus allosteric models of IKs activation by comparing simulations with experimental steady-state and transient activation kinetics, voltage-sensor fluorescence from channels with two or four activatable domains, and limiting slope currents at negative potentials. Sequential Hodgkin–Huxley-type models approximately describe IKs currents but cannot explain an activation delay in channels with only one activatable subunit or the hyperpolarizing shift in the conductance–voltage relationship with more activatable voltage sensors. Incorporating two voltage sensor activation steps in sequential models and a concerted step in opening via rates derived from fluorescence measurements improves models but does not resolve fundamental differences with experimental data. Limiting slope current data that show the opening of channels at negative potentials and very low open probability are better simulated using allosteric models of activation with one transition per voltage sensor, which implies that movement of all four sensors is not required for IKs conductance. Tiered allosteric models with two activating transitions per voltage sensor can fully account for IKs current and fluorescence activation kinetics in constructs with different numbers of activatable voltage sensors.

In cardiomyocytes, the KCNQ1/KCNE1 channel complex mediates the slow delayed-rectifier current (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause long QT syndrome (LQTS), a syndrome with a prolonged QT interval on the ECG, which increases the risk of ventricular arrhythmia and sudden cardiac death. One potential therapeutical intervention for LQTS is based on targeting IKs channels to restore channel function and/or the physiological QT interval. Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels and activate IKs channels by binding to two different sites, one in the voltage sensor domain – which shifts the voltage dependence to more negative voltages – and the other in the pore domain – which increases the maximal conductance of the channels (Gmax). However, the mechanism by which PUFAs increase the Gmax of the IKs channels is still poorly understood. In addition, it is unclear why IKs channels have a very small single-channel conductance and a low open probability or whether PUFAs affect any of these properties of IKs channels. Our results suggest that the selectivity filter in KCNQ1 is normally unstable, contributing to the low open probability, and that the PUFA-induced increase in Gmax is caused by a stabilization of the selectivity filter in an open-conductive state.

Polyunsaturated fatty acids (PUFAs) are potent activators of KCNQ1 channels, found in smooth muscle cells and cardiomyocytes. In the latter, the channel forms the KCNQ1/KCNE1 complex, mediating the slow delayed-rectifier currents (IKs), pivotal during the repolarization phase of the ventricular action potential. Mutations in IKs cause Long QT Syndrome (LQT), which increases the risk of sudden cardiac death and ventricular arrhythmia. One possible therapeutic intervention would be based on specifically targeting IKs currents to restore the channel function and the physiological QT interval. PUFAs are ideal candidates as they activate IKs current by shifting the voltage dependence of opening toward negative voltages and by increasing the maximum conductance of the channel (Gmax). PUFAs mechanism of action rely on two different binding sites, one at the voltage sensor domain - which shifts the voltage dependence - and the other at the pore region of the channel - which increases the Gmax. However, the mechanism by which PUFAs increase the Gmax of the channels is still poorly understood. In this study, we propose that the increase in the channel currents is caused by a complex mechanism of several residues in the external mouth of the pore, including K326, P320, and the two tryptophans of the conserved aromatic ring cuff. Our investigations showed that by mutating some of these residues, the effect of PUFAs on Gmax is reduced, describing a complex mechanism involving a wide network of residues in the Gmax effect of PUFAs.

The IKs current formed by the co-assembly of KCNQ1 and KCNE1 plays an important role in cardiac repolarization. Loss-of-function mutations in KCNQ1 and KCNE1 are linked to LQT syndrome. Mefenamic acid (Mef) an NSAID, and DIDS a stilbene, are known to enhance IKs currents. The present study explores in detail the role of previously identified K41-KCNE1 in Mef action as well as uncovering the KCNQ1/KCNE1 binding site(s) of Mef and DIDS. In silico experiments of drug docking on a model of IKs channel and molecular dynamic (MD) simulations were used to map critical KCNQ1 and KCNE1 residues, followed by site-directed mutagenesis and functional analysis using whole cell patch clamp. Docking procedures and MD simulations show that Mef and DIDS bind between extracellular KCNE1 residues, specifically to the subunits of the external S6 helix; neighbouring S1; and the pore turret of the opposing subunit. Mef transforms the slowly activating WT IKs current (4:4 EQ) into a current with an almost linear waveform, which causes a left shift in activation V1/2 and inhibits tail current decay at −40 mV. These effects are prevented by the K41C-KCNE1 mutation in a stoichiometric manner. MD simulations and mutagenesis scanning identified three critical residues for Mef binding: W323-KCNQ1; Y46-KCNE1; and K41-KCNE1 in which Mef effects are greatly diminished. The effect of DIDS on WT-EQ are less pronounced than Mef, and K41C-KCNE1 was unable to fully prevent effects of DIDS. Our results show that both activators bind in the same general channel region but depend on different critical residues. These findings provide insights into a common drug-induced binding site, suggesting that the chemical structure of either compound could serve as a starting point for the development of IKs agonists.

The cardiac IKs ion channel comprises KCNQ1, calmodulin, and KCNE1 in a dodecameric complex which provides a repolarizing current reserve at higher heart rates and protects from arrhythmia syndromes that cause fainting and sudden death. Pharmacological activators of IKs are therefore of interest both scientifically and therapeutically for treatment of IKs loss-of-function disorders. One group of chemical activators are only active in the presence of the accessory KCNE1 subunit and here we investigate this phenomenon using molecular modeling techniques and mutagenesis scanning in mammalian cells. A generalized activator binding pocket is formed extracellularly by KCNE1, the domain-swapped S1 helices of one KCNQ1 subunit and the pore/turret region made up of two other KCNQ1 subunits. A few residues, including K41, A44 and Y46 in KCNE1, W323 in the KCNQ1 pore, and Y148 in the KCNQ1 S1 domain, appear critical for the binding of structurally diverse molecules, but in addition, molecular modeling studies suggest that induced fit by structurally different molecules underlies the generalized nature of the binding pocket. Activation of IKs is enhanced by stabilization of the KCNQ1-S1/KCNE1/pore complex, which ultimately slows deactivation of the current, and promotes outward current summation at higher pulse rates. Our results provide a mechanistic explanation of enhanced IKs currents by these activator compounds and provide a map for future design of more potent therapeutically useful molecules.

KCNQ1 voltage-gated K+ channels are involved in a wide variety of fundamental physiological processes and exhibit the unique feature of being markedly inhibited by external K+. Despite the potential role of this regulatory mechanism in distinct physiological and pathological processes, its exact underpinnings are not well understood. In this study, using extensive mutagenesis, molecular dynamics simulations, and single-channel recordings, we delineate the molecular mechanism of KCNQ1 modulation by external K+. First, we demonstrate the involvement of the selectivity filter in the external K+ sensitivity of the channel. Then, we show that external K+ binds to the vacant outermost ion coordination site of the selectivity filter inducing a diminution in the unitary conductance of the channel. The larger reduction in the unitary conductance compared to whole-cell currents suggests an additional modulatory effect of external K+ on the channel. Further, we show that the external K+ sensitivity of the heteromeric KCNQ1/KCNE complexes depends on the type of associated KCNE subunits.

The KCNQ1 ion channel plays critical physiological roles in electrical excitability and K + recycling in organs including the heart, brain, and gut. Loss of function is relatively common and can cause sudden arrhythmic death, sudden infant death, epilepsy and deafness. Here, we report cryogenic electron microscopic (cryo-EM) structures of Xenopus KCNQ1 bound to Ca 2+ /Calmodulin, with and without the KCNQ1 channel activator, ML277. A single binding site for ML277 was identified, localized to a pocket lined by the S4-S5 linker, S5 and S6 helices of two separate subunits. Several pocket residues are not conserved in other KCNQ isoforms, explaining specificity. MD simulations and point mutations support this binding location for ML277 in open and closed channels and reveal that prevention of inactivation is an important component of the activator effect. Our work provides direction for therapeutic intervention targeting KCNQ1 loss of function pathologies including long QT interval syndrome and seizures.

G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important physiological roles in various organs. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited GIRK mutants. By the two-electrode voltage-clamp analysis of GIRK mutants heterologously expressed in Xenopus oocytes, we observed that Kir3.2 G156S permeates Li+ better than Rb+ , while T154del or L173R of Kir3.2 and T158A of Kir3.4 permeate Rb+ better than Li+ , suggesting a unique conformational change in the G156S mutant. Applications of blockers of the selectivity filter (SF) pathway, Ba2+ or Tertiapin-Q (TPN-Q), remarkably increased the Li+ -selectivity of Kir3.2 G156S but did not alter those of the other mutants. In single-channel recordings of Kir3.2 G156S expressed in mouse fibroblasts, two types of events were observed, one attributable to a TPN-Q-sensitive K+ current and the second a TPN-Q-resistant Li+ current. The results show that a novel Li+ -permeable and blocker-resistant pathway exists in G156S in addition to the SF pathway. Mutations in the pore helix, S148F and T151A also induced high Li+ permeation. Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations. KEY POINTS: G-protein-gated inwardly rectifying K+ (GIRK; Kir3.x) channels play important roles in controlling excitation of cells in various organs, such as the brain and the heart. Some of the disease-associated mutations of GIRK channels are known to induce loss of K+ selectivity but their structural changes remain unclear. In this study, we investigated the mechanisms underlying the abnormal ion selectivity of inherited mutants of Kir3.2 and Kir3.4. Here we show that a novel Na+ , Li+ -permeable and blocker-resistant pathway exists in an inherited mutant, Kir3.2 G156S, in addition to the conventional ion conducting pathway formed by the selectivity filter (SF). Our results demonstrate that the mechanism underlying the loss of K+ selectivity of Kir3.2 G156S involves formation of a novel ion permeation pathway besides the SF pathway, which allows permeation of various species of cations.

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell-derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

The pairing of KCNQ1 and KCNE1 in the heart, together mediates the slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization. The nonsteroidal-anti-inflammatory drug, mefenamic acid, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid's effect on IKs. Using whole-cell patch-clamp, we show that mefenamic acid enhances IKsactivity in a dose-dependent manner by changing the slowly activating IKs current into an almost linear current with instantaneous onset which, can in turn, be inhibited by the IKs blocker, HMR1556. Both single channel studies which reveal no changes in the maximum conductance, and whole-cell studies which reveal a dramatically altered G-V relationship despite increasingly longer interpulse intervals, suggest that mefenamic acid decreases the voltage sensitivity of the IKschannel and causes a hyperpolarizing shift in the voltage-dependence of channel activation. Since the stoichiometry of KCNQ1 to KCNE1 subunits has the potential to vary up to a ratio of 4:4, it was important to examine the effect of other IKs stoichiometries on the action of mefenamic acid. No effect on KCNQ1 alone was found and the degree of current activation was dependent on the number of KCNE1 subunits present, which suggested a binding site on KCNE1 may exist. Consistent with this, mutational analysis revealed an extracellular site of significance on KCNE1. The charge on residue Lys41 and, potentially other surrounding residues, are critical for the activating effect of mefenamic acid on IKs. Overall, unlike most other IKs activators which have little effect on IKs channels with increasingly saturated stoichiometries,wehave shown that mefenamic acid is able to enhance all Iks channel complexes containing different KCNQ1:KCNE1 stoichiometries which suggest it may represent a promising therapeutic approach to treating long QT syndromes.