Channel KCNE1 Research Articles

Dual effects of mefenamic acid on the IKs molecular complex.

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. 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. 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. 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 endocannabinoid ARA-S facilitates the activation of cardiac Kv7.1/KCNE1 channels from different species

ABSTRACT The endogenous endocannabinoid-like compound N-arachidonoyl-L-serine (ARA-S) facilitates activation of the human Kv7.1/KCNE1 channel and shortens a prolonged action potential duration and QT interval in guinea pig hearts. Hence, ARA-S is interesting to study further in cardiac models to explore the functional impact of such Kv7.1/KCNE1-mediated effects. To guide which animal models would be suitable for assessing ARA-S effects, and to aid interpretation of findings in different experimental models, it is useful to know whether Kv7.1/KCNE1 channels from relevant species respond similarly to ARA-S. To this end, we used the two-electrode voltage clamp technique to compare the effects of ARA-S on Kv7.1/KCNE1 channels from guinea pig, rabbit, and human Kv7.1/KCNE1, when expressed in Xenopus laevis oocytes. We found that the activation of Kv7.1/KCNE1 channels from all tested species was facilitated by ARA-S, seen as a concentration-dependent shift in the voltage-dependence of channel opening and increase in current amplitude and conductance over a broad voltage range. The rabbit channel displayed quantitatively similar effects as the human channel, whereas the guinea pig channel responded with more prominent increase in current amplitude and maximal conductance. This study suggests that rabbit and guinea pig models are both suitable for studying ARA-S effects mediated via Kv7.1/KCNE1.

The fully activated open state of KCNQ1 controls the cardiac "fight-or-flight" response.

The cardiac KCNQ1 + KCNE1 (IKs) channel regulates heart rhythm under both normal and stress conditions. Under stress, the β-adrenergic stimulation elevates the intracellular cyclic adenosine monophosphate (cAMP) level, leading to KCNQ1 phosphorylation by protein kinase A and increased IKs, which shortens action potentials to adapt to accelerated heart rate. An impaired response to the β-adrenergic stimulation due to KCNQ1 mutations is associated with the occurrence of a lethal congenital long QT syndrome (type 1, also known as LQT1). However, the underlying mechanism of β-adrenergic stimulation of IKs remains unclear, impeding the development of new therapeutics. Here, we find that the unique properties of KCNQ1 channel gating with two distinct open states are key to this mechanism. KCNQ1's fully activated open (AO) state is more sensitive to cAMP than its intermediate open state. By enhancing the AO state occupancy, the small molecules ML277 and C28 are found to effectively enhance the cAMP sensitivity of the KCNQ1 channel, independent of KCNE1 association. This finding of enhancing AO state occupancy leads to a potential novel strategy to rescue the response of IKs to β-adrenergic stimulation in LQT1 mutants. The success of this approach is demonstrated in cardiac myocytes and also in a high-risk LQT1 mutation. In conclusion, the present study not only uncovers the key role of the AO state in IKs channel phosphorylation, but also provides a target for antiarrhythmic strategy.

The endocannabinoid ARA-S acts as an endogenous rescuing factor of Kv7.1/KCNE1 carrying Long QT syndrome mutations and paves the way for drug development strategies

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): European Union’s Horizon 2020 research and innovation program Long QT Syndrome (LQTS) is a cardiac arrhythmia characterized by delayed ventricular repolarization. LQTS occurs when there are loss-of-function mutations in genes encoding for voltage-gated channels and their beta subunits that contribute to the cardiac action potential. Among them, the most mutated channel is Kv7.1/KCNE1, which encodes for the slowly activating component of the delayed rectifier K+ current (IKs). Families with inherited mutations in the Kv7.1/KCNE1 exhibit inconsistent correlations between genotypes and phenotypes making the clinical management of LQTS challenging. A possible strategy to help guide clinical approaches involves exploring endogenous compounds capable of modulating the Kv7.1/KCNE1 channel function, acting as potential disease modifiers. Using the two-electrode voltage clamp technique, we previously found that the endogenous endocannabinoid-like compound ARA-S, which has an arachidonic acid tail and a serine head group, facilitates activation of the wild-type Kv7.1/KCNE1 channel expressed in Xenopus oocytes. This is seen as a left shift in the voltage dependence of channel opening and an increase in conductance. Moreover, we showed that ARA-S restores a physiological QT interval in isolated guinea pig hearts suffering from drug-induced LQTS. Hence, ARA-S is an interesting endogenous Kv7.1/KCNE1 channel modulator that may influence disease severity. However, it is uncertain whether ARA-S also activates the Kv7.1/KCNE1 when it carries LQTS-associated mutations. Therefore, the current study involves studying the effect of ARA-S on mutated Kv7.1/KCNE1 channels using two-electrode voltage clamp electrophysiology. We find that ARA-S facilitates the activation of all tested LQTS mutants. However, the magnitude of the effect varies among them, suggesting that the compound´s effect is dependent on the genetic background. To understand why ARA-S has different effects, we use computational simulation approaches to study how mutations in the putative ARA-S binding sites change ARA-S efficacy. This understanding aids in developing new substances that can activate different mutations, by, for example, modifying the ARA-S tail to enhance its efficacy. We also try to understand the concentration of ARA-S required to restore a wild-type-like function of each mutant, considering the mutant's intrinsic behavior and the apparent affinity of ARA-S for the specific mutant. Altogether, this work finds ARA-S to be an efficient activator of LQTS-associated mutant Kv7.1/KCNE1 channels and is a first step towards understanding how the genetic background determines the effect of this endogenous modulator.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes.

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.

A generic binding pocket for small molecule IKs activators at the extracellular inter-subunit interface of KCNQ1 and KCNE1 channel complexes

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.

Subtype-specific modulation of human KV 7 channels by the anticonvulsant cannabidiol through a lipid-exposed pore-domain site.

Cannabidiol (CBD) is used clinically as an anticonvulsant. Its precise mechanism of action has remained unclear. CBD was recently demonstrated to enhance the activity of the neuronal KV 7.2/7.3 channel, which may be one important contributor to CBD anticonvulsant effect. Curiously, CBD inhibits the closely related cardiac KV 7.1/KCNE1 channel. Whether and how CBD affects other KV 7 subtypes remains uninvestigated and the CBD interaction sites mediating these diverse effects remain unknown. Here, we used electrophysiology, molecular dynamics simulations, molecular docking and site-directed mutagenesis to address these questions. We found that CBD modulates the activity of all human KV 7 subtypes and that the effects are subtype dependent. CBD enhanced the activity of KV 7.2-7.5 subtypes, seen as a V50 shift towards more negative voltages or increased maximum conductance. In contrast, CBD inhibited the KV 7.1 and KV 7.1/KCNE1 channels, seen as a V50 shift towards more positive voltages and reduced conductance. In KV 7.2 and KV 7.4, we propose a CBD interaction site at the subunit interface in the pore domain that overlaps with the interaction site of other compounds, notably the anticonvulsant retigabine. However, CBD relies on other residues for its effects than the conserved tryptophan that is critical for retigabine effects. We propose a similar, though not identical CBD site in KV 7.1, with a non-conserved phenylalanine being important. We identify novel targets of CBD, contributing to a better understanding of CBD clinical effects and provide mechanistic insights into how CBD modulates different KV 7 subtypes.

Long-QT mutations in KCNE1 modulate the 17β-estradiol response of Kv7.1/KCNE1

Estradiol (17[Formula: see text]-E2) is implicated in higher arrhythmia risk of women with congenital or acquired long-QT syndrome (LQTS) compared to men. However, the underlying mechanisms remain poorly understood, and little is known about the impact of LQTS-associated mutations. We show that 17[Formula: see text]-E2 inhibits the human cardiac Kv7.1/KCNE1 channel expressed in Xenopus oocytes. We find that the 17[Formula: see text]-E2 effect depends on the Kv7.1 to KCNE1 stoichiometry, and we reveal a critical function of the KCNE1 carboxyl terminus for the effect. LQTS-associated mutations in the KCNE1 carboxyl terminus show a range of responses to 17[Formula: see text]-E2, from a wild-type like response to impaired or abolished response. Together, this study increases our understanding of the mechanistic basis for 17[Formula: see text]-E2 inhibition of Kv7.1/KCNE1 and demonstrates mutation-dependent responses to 17[Formula: see text]-E2. These findings suggest that the 17[Formula: see text]-E2 effect on Kv7.1/KCNE1 might contribute to the higher arrhythmia risk of women, particularly in carriers with specific LQTS-associated mutations.

Endocannabinoids enhance hKV7.1/KCNE1 channel function and shorten the cardiac action potential and QT interval.

Genotype-positive patients who suffer from the cardiac channelopathy Long QT Syndrome (LQTS) may display a spectrum of clinical phenotypes, with often unknown causes. Therefore, there is a need to identify factors influencing disease severity to move towards an individualized clinical management of LQTS. One possible factor influencing the disease phenotype is the endocannabinoid system, which has emerged as a modulator of cardiovascular function. In this study, we aim to elucidate whether endocannabinoids target the cardiac voltage-gated potassium channel KV7.1/KCNE1, which is the most frequently mutated ion channel in LQTS. We used two-electrode voltage clamp, molecular dynamics simulations and the E4031 drug-induced LQT2 model of ex-vivo guinea pig hearts. We found a set of endocannabinoids that facilitate channel activation, seen as a shifted voltage-dependence of channel opening and increased overall current amplitude and conductance. We propose that negatively charged endocannabinoids interact with known lipid binding sites at positively charged amino acids on the channel, providing structural insights into why only specific endocannabinoids modulate KV7.1/KCNE1. Using the endocannabinoid ARA-S as a prototype, we show that the effect is not dependent on the KCNE1 subunit or the phosphorylation state of the channel. In guinea pig hearts, ARA-S was found to reverse the E4031-prolonged action potential duration and QT interval. We consider the endocannabinoids as an interesting class of hKV7.1/KCNE1 channel modulators with putative protective effects in LQTS contexts. ERC (No. 850622), Canadian Institutes of Health Research, Canada Research Chairs and Compute Canada, Swedish National Infrastructure for Computing.

The endocannabinoid ARA-S displays variably activating effects on Kv7.1/KCNE1 carrying long QT syndrome mutations.

The cardiac arrhythmia long QT syndrome (LQTS) is characterized by a delayed ventricular repolarization. The most common cause of inherited LQTS is loss-of-function mutations in the KV7.1/KCNE1 channel. Therefore, the KV7.1/KCNE1 channel is an interesting target for developing new antiarrhythmic drugs, which ideally would restore the function of mutated KV7.1/KCNE1 channels. In this study, we evaluate the endogenous endocannabinoid-like compound ARA-S as an activator of the human KV7.1/KCNE1 channel carrying diverse loss-of-function mutations linked to LQTS. Using the two-electrode voltage clamp technique, we have previously found ARA-S to facilitate activation of the wild type KV7.1/KCNE1 channel expressed in Xenopus oocytes, seen as a left-shift in voltage dependence of channel opening and a conductance increase. Moreover, we have previously shown that ARA-S restores a physiological QT interval in isolated guinea pig hearts suffering from drug-induced LQTS. Hence, ARA-S is an interesting endogenous KV7.1/KCNE1 channel modulator which may inspire drug development. Now, using the same technique on LQTS-mutated KV7.1/KCNE1 channels, we find that ARA-S facilitates activation of all tested mutants, but the magnitude of the effect varies among them. For instance, the effect of ARA-S is reduced on the mutant I204F but increased on the mutant A370V, compared to the effect on the wild-type channel. This suggests that the effect of the compound is dependent on the genetic background. To better understand its impact, we group the tested mutants based on their response to ARA-S and factors such as whether they are localized to putative ARA-S binding sites or other interesting sites, such as PIP2 sites. Altogether, this work finds ARA-S as an efficient activator of LQTS-associated mutant KV7.1/KCNE1 channels and is a first step towards understanding how the genetic background determines the effect of this modulator.

Human-induced pluripotent stem cell-derived cardiomyocytes to study PUFA analogues as a potential treatment of long QT syndrome.

Long QT syndrome (LQTS) is an inheritable ventricular arrhythmia that predisposes patients to torsades de pointes and sudden cardiac death. Mutations in voltage-gated ion channels (VGC) and their regulatory β subunits involved in the cardiac action potential (AP) generate LQTS by prolonging the QT interval of the electrocardiogram. 15 types of LQTS has been described depending on the underlying gene mutated. LQT1 and LQT5 are described as loss-of-function mutations in genes that encode Kv7.1 (alpha pore-forming subunit) and its β-regulatory subunit KCNE1, respectively. Kv7.1/KCNE1 channels elicit one of the main repolarization currents (IKs) involved in the cardiac AP. Therefore, Kv7.1/KCNE1 channel-specific activators might be a potential treatment for LQTS. Polyunsaturated fatty acid (PUFA) analogues can broadly modulate cardiac VGC when applied on isolated channels in a heterologous system. In the present study, we measured the effects of these four compounds, Linoleoyl-taurine (LIN-Tau), docosahexaenoyl-Tau (DHA-Tau), Linoleoyl-Glycine (LIN-Gly), and DHA-Gly, on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). DHA-analogues shortened the hiPSC-CMs AP duration (APD), while LIN-analogues prolonged hiPSC-CMs APD. DHA-analogues were also tested in isogenic hiPSC-CMs lines for two KCNQ1 (Kv7.1 gene) mutations that cause LQT1, R190Q, and G269S. R190Q is an autosomal dominant missense mutation characterized by a 70-80 % reduction of IKs because of a dominant negative traffic defect. Moreover, we also analyzed DHA-analogues in a heterozygous missense KCNQ1 mutation, G269S. G269S mutation shows a net effect of less than 50% reduction of IKs current (haploinsufficiency). We here use a combination of hiPSC-CM monolayers with a high-resolution, all-optical electrophysiology system for screening the anti-arrhythmic properties of diverse PUFA analogues. Moreover, the well-characterized hiPSC-CMs mutants show us whether PUFA analogues might be a potential treatment approach for more than one type of LQT1.

Endocannabinoid-like compounds as modulators of the cardiac kv7.1/KCNE1 channel

The cardiac voltage-gated potassium channel Kv7.1/KCNE1 has an important role in the ventricular cardiac action potential, where it contributes to the repolarization phase. Loss-of-function mutations in the genes encoding for Kv7.1/KCNE1 have been functionally linked to the arrythmia long QT syndrome. Up to 30% of individuals with some of the mentioned inherited mutations are not well protected from suffering cardiac events, because there is no specific medication targeting the Kv7.1/KCNE1 channel.

Effect of Channel Assembly (KCNQ1 or KCNQ1 + KCNE1) on the Response of Zebrafish IKs Current to IKs Inhibitors and Activators.

In cardiac myocytes, the slow component of the delayed rectifier K+ current (IKs) ensures repolarization of action potential during beta-adrenergic activation or when other repolarizing K+ currents fail. As a key factor of cardiac repolarization, IKs should be present in model species used for cardiovascular drug screening, preferably with pharmacological characteristics similar to those of the human IKs. To this end, we investigated the effects of inhibitors and activators of the IKs on KCNQ1 and KCNQ1 + KCNE1 channels of the zebrafish, an important model species, in Chinese hamster ovary cells. Inhibitors of IKs, chromanol 293B and HMR-1556, inhibited zebrafish IKs channels with approximately similar potency as that of mammalian IKs. Chromanol 293B concentration for half-maximal inhibition (IC50) of zebrafish IKs was at 13.1 ± 5.8 and 13.4 ± 2.8 µM for KCNQ1 and KCNQ1+KCNE1 channels, respectively. HMR-1556 was a more potent inhibitor of zebrafish IKs channels with IC50 = 0.1 ± 0.1 µM and 1.5 ± 0.8 µM for KCNQ1 and KCNQ1 + KCNE1 channels, respectively. R-L3 and mefenamic acid, generally identified as IKs activators, both inhibited zebrafish IKs. R-L3 almost completely inhibited the current generated by KCNQ1 and KCNQ1 + KCNE1 channels with similar potency (IC50 1.1 ± 0.4 and 1.0 ± 0.4 µM, respectively). Mefenamic acid partially blocked zebrafish KCNQ1 (IC50 = 9.5 ± 4.8 µM) and completely blocked KCNQ1 + KCNE1 channels (IC50 = 3.3 ± 1.8 µM). Although zebrafish IKs channels respond to IKs inhibitors in the same way as mammalian IKs channels, their response to activators is atypical, probably because of the differences in the binding domain of KCNE1 to KCNQ1. Therefore, care must be taken when translating the results from zebrafish to humans.

COVID-19 Drugs Chloroquine and Hydroxychloroquine, but Not Azithromycin and Remdesivir, Block hERG Potassium Channels.

Drug-induced long QT syndrome (LQTS) is an established cardiac side effect of a wide range of medications and represents a significant concern for drug safety. The rapidly and slowly activating delayed rectifier K+ currents, mediated by channels encoded by the human ether-a-go-go-related gene (hERG) and KCNQ1 + KCNE1, respectively, are two main currents responsible for ventricular repolarization. The common cause for drugs to induce LQTS is through impairing the hERG channel. For the recent emergence of COVID-19, caused by severe acute respiratory syndrome coronavirus 2, several drugs have been investigated as potential therapies; however, there are concerns about their QT prolongation risk. Here, we studied the effects of chloroquine, hydroxychloroquine, azithromycin, and remdesivir on hERG channels. Our results showed that although chloroquine acutely blocked hERG current (IhERG), with an IC50 of 3.0 µM, hydroxychloroquine acutely blocked IhERG 8-fold less potently, with an IC50 of 23.4 µM. Azithromycin and remdesivir did not acutely affect IhERG When these drugs were added at 10 µM to the cell culture medium for 24 hours, remdesivir increased IhERG by 2-fold, which was associated with an increased mature hERG channel expression. In addition, these four drugs did not acutely or chronically affect KCNQ1 + KCNE1 channels. Our data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns. SIGNIFICANCE STATEMENT: This work demonstrates that, among off-label potential COVID-19 treatment drugs chloroquine, hydroxychloroquine, azithromycin, and remdesivir, the former two drugs block hERG potassium channels, whereas the latter two drugs do not. All four drugs do not affect KCNQ1 + KCNE1. As hERG and KCNQ1 + KCNE1 are two main K+ channels responsible for ventricular repolarization, and most drugs that induce long QT syndrome (LQTS) do so by impairing hERG channels, these data provide insight into COVID-19 drug-associated LQTS and cardiac safety concerns.

Gating and Regulation of KCNQ1 and KCNQ1 + KCNE1 Channel Complexes.

The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its β-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1−4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

Physical and functional interaction sites in cytoplasmic domains of KCNQ1 and KCNE1 channel subunits.

The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and β-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and β-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352-374 and KCNE1 residues 70-81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region.NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.

IKs ion-channel pore conductance can result from individual voltage sensor movements

The IKs current has an established role in cardiac action potential repolarization, and provides a repolarization reserve at times of stress. The underlying channels are formed from tetramers of KCNQ1 along with one to four KCNE1 accessory subunits, but how these components together gate the IKs complex to open the pore is controversial. Currently, either a concerted movement involving all four subunits of the tetramer or allosteric regulation of open probability through voltage-dependent subunit activation is thought to precede opening. Here, by using the E160R mutation in KCNQ1 or the F57W mutation in KCNE1 to prevent or impede, respectively, voltage sensors from moving into activated conformations, we demonstrate that a concerted transition of all four subunits after voltage sensor activation is not required for the opening of IKs channels. Tracking voltage sensor movement, via [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) modification and fluorescence recordings, shows that E160R-containing voltage sensors do not translocate upon depolarization. E160R, when expressed in all four KCNQ1 subunits, is nonconducting, but if one, two, or three voltage sensors contain the E160R mutation, whole-cell and single-channel currents are still observed in both the presence and absence of KCNE1, and average conductance is reduced proportional to the number of E160R voltage sensors. The data suggest that KCNQ1 + KCNE1 channels gate like KCNQ1 alone. A model of independent voltage sensors directly coupled to open states can simulate experimental changes in IKs current kinetics, including the nonlinear depolarization of the conductance-voltage (G-V) relationship, and tail current acceleration as the number of nonactivatable E160R subunits is increased.

Modulation of Kv7.1/KCNE1 Channel Activity by Navβ1

KV7.1 is a voltage-gated potassium channel encoded by the gene KCNQ1. This channel is found in several organs, including the heart and the brain. In the mammalian heart, KV7.1 assembles with KCNE1 forming a channel complex that is critically important for ventricular repolarization. The activity of the KV7.1/KCNE1 complex results in the delayed rectifier current known as IKs. The voltage-gated sodium channel β type 1 subunit (NaVβ1) is also expressed in the mammalian heart. Mutations in this protein have been implicated in disorders such as epilepsy and cardiopathies, including Brugada syndrome and atrial fibrillation. The NaVβ1 subunit modulates the activity, trafficking, and expression of NaV channels. It has been shown that NaVβ1 also modulates channels from the KV1 and KV4 families, and KV7.2 channels. Preliminary data from our laboratory show that NaVβ1 modulates the activity of KV7.1. When expressed in Xenopus oocytes, KV7.1 channel-mediated K+-currents display a mild slow inactivation. These channels recover from inactivation during deactivation driven at negative voltage, displaying a “hook” like kinetics. In the presence of NaVβ1, the inactivation is remarkably enhanced, producing a strong resurgent conductance during deactivation at negative potentials. Furthermore, the deactivation rate is decreased, while activation remains seemingly unaltered. Since KV7.1, KCNE1 and NaVβ1 are expressed in the heart, we decided to investigate whether NaVβ1 could modulate KV7.1/KCNE1 activity. We found that expressing KCNE1 subunit seems to override the NaVβ1 subunit effect on KV7.1 channels. However, decreasing the relative expression of KCNE1 resulted in K+-currents that displayed features of the activity of both type of complexes. We therefore conclude that NaVβ1 can modulate the KV7.1/KCNE1 complex activity. Our work suggests that, in the heart, the interaction of KCNE1 with KV7.1 has evolved to avoid the effect of NaVβ1 on the channel, so preventing its inactivation.

NMR resonance assignments and secondary structure of a mutant form of the human KCNE1 channel accessory protein that exhibits KCNE3-like function.

KCNQ1 (Q1) is a voltage-gated potassium channel that is modulated by members of the KCNE family, the best-characterized being KCNE1 (E1) and KCNE3 (E3). The Q1/E1 complex generates a channel with delayed activation and increased conductance. This complex is expressed in cardiomyocytes where it provides the IKs current that is critical for the repolarization phase of the cardiac action potential. The Q1/E3 complex, on the other hand, is expressed in epithelial cells of the colon and stomach, where it serves as a constitutively active leak channel to help maintain water and ion homeostasis. Studies show the single transmembrane segments (TMS) present in both E1 and E3 are essential to their distinct functions. More specifically, residues FTL located near the middle of the E1 TMS are essential for the delayed activation of Q1, while the corresponding TVG sites in E3 are critical for constitutive activation of the channel. Swapping these three residues leads to the switching of the functional properties for both Q1/E1FTL→TVG and Q1/E3TVG→FTL complexes. This work details the backbone assignments and chemical shifts for the E1FTL→TVG mutant, as determined using a suite of 3D NMR experiments along with specific and inverse amino acid isotopic labeling. The completed assignments can be used, in conjunction with other NMR experiments, to generate a 3D structure of E1FTL→TVG. The results of TALOS-N analysis of the chemical shifts are reported here. The E1FTL→TVG structure will be compared to the already available E1 and E3 structures to determine the roles that their TMS triplet motifs play in each protein to dictate their distinct channel-modulatory functions.

Mechanisms Underlying the Dual Effect of Polyunsaturated Fatty Acid Analogs on Kv7.1

Polyunsaturated fatty acid (PUFA) analogs represent a new class of potential anti-arrhythmic KV7.1 and KV7.1+KCNE1 channel activators. In this study, we describe dual independent activating effects of negatively charged PUFA analogs on KV7.1 and KV7.1+KCNE1 that are dependent on discrete channel motifs. PUFA analogs are critically dependent on K326 in S6 of KV7.1 to increase the maximum conductance and critically dependent on specific S4 arginines in KV7.1 to shift the voltage dependence of channel opening toward negative voltages. Our findings provide insights into how KV7.1+KCNE1 activators may interact electrostatically both with the pore domain and the voltage-sensing domain to augment channel activity. We believe that the molecular understanding of how PUFA analogs induce dual independent activating effects is an important step toward the development of effective anti-arrhythmic drugs that target KV7.1 channels.