Wild-type hERG Research Articles

Rescue of expression and function of long QT syndrome-causing mutant hERG channels by enhancing channel stability in the plasma membrane

The human ether-g-go-go-related gene (hERG) encodes the Kv11.1 (or hERG) channel that conducts the rapidly activating delayed rectifier potassium current (IKr). Naturally occurring mutations in hERG impair the channel function and cause long QT syndrome type 2 (LQT2). Many missense hERG mutations lead to a lack of channel expression on the cell surface, representing a major mechanism for the loss-of-function of mutant channels. While it is generally thought that a trafficking defect underlies the lack of channel expression on the cell surface, in the present study, we demonstrate that the trafficking defective mutant hERG G601S can reach the plasma membrane but is unstable and quickly degrades, which is akin to Wild Type (WT) hERG channels under low K+ conditions. We previously showed that Serine (S) residue at 624 in the innermost position of the selectivity filter of hERG is involved in hERG membrane stability such that substitution of Serine 624 with Threonine (S624T) enhances hERG stability and renders hERG insensitive to low K+ culture. Here, we report that the intragenic addition of S624T substitution to trafficking defective hERG mutants G601S, N470D and P596R led to a complete rescue of the function of these otherwise loss-of-function mutant channels to a level similar to the WT channel, representing the most effective rescue means for the function of mutant hERG channels. These findings not only provide novel insights into hERG mutation-mediated channel dysfunction, but also point to the critical role of S624 in hERG stability on the plasma membrane.

A Novel KCNH2 S981fs Mutation Identified by Whole-Exome Sequencing Is Associated with Type 2 Long QT Syndrome.

KCNH2 loss-of-function mutations cause long QT syndrome type 2 (LQT2), an inherited cardiac disorder associated with life-threatening ventricular arrhythmia. Through whole-exome sequencing, we discovered a novel AGCGACAC deletion (S981fs) in the hERG gene of an LQT2 patient. Using a heterologous expression system and patch clamping, we found that the mutant K channel had reduced cell surface expression and lower current amplitude compared to the wild type. However, functional expression was restored by lowering temperature and using potassium channel inhibitors or openers (E4031, cisapride, nicorandil). Co-immunoprecipitation experiments confirmed the assembly of mutant proteins with wild-type hERG. Confocal imaging showed decreased hERG distribution on the cell membrane in cells expressing S981fs. Notably, treatment with G418 significantly increased hERG current in wild-type/S981fs heterozygotes. In conclusion, our study identifies a novel hERG mutation leading to impaired Kv11.1 function due to trafficking and nonsense-mediated RNA decay defects. These findings shed light on the mechanisms underlying LQT2 and offer potential therapeutic avenues.

Differential Effects of Remdesivir and Lumacaftor on Homomeric and Heteromeric hERG Channels.

The human ether-a-go-go-related gene (hERG) encodes for the pore-forming subunit of the channel that conducts the rapidly activating delayed K+ current (IKr) in the heart. The hERG channel is important for cardiac repolarization, and reduction of its expression in the plasma membrane due to mutations causes long QT syndrome type 2 (LQT2). As such, promoting hERG membrane expression is a strategy to rescue mutant channel function. In the present study, we applied patch clamp, western blots, immunocytochemistry, and quantitative reverse transcription polymerase chain reaction techniques to investigate the rescue effects of two drugs, remdesivir and lumacaftor, on trafficking-defective mutant hERG channels. As our group has recently reported that the antiviral drug remdesivir increases wild-type (WT) hERG current and surface expression, we studied the effects of remdesivir on trafficking-defective LQT2-causing hERG mutants G601S and R582C expressed in HEK293 cells. We also investigated the effects of lumacaftor, a drug used to treat cystic fibrosis, that promotes CFTR protein trafficking and has been shown to rescue membrane expression of some hERG mutations. Our results show that neither remdesivir nor lumacaftor rescued the current or cell-surface expression of homomeric mutants G601S and R582C. However, remdesivir decreased while lumacaftor increased the current and cell-surface expression of heteromeric channels formed by WT hERG and mutant G601S or R582C hERG. We concluded that drugs can differentially affect homomeric WT and heteromeric WT+G601S (or WT+R582C) hERG channels. These findings extend our understanding of drug-channel interaction and may have clinical implications for patients with hERG mutations. SIGNIFICANCE STATEMENT: Various naturally occurring mutations in a cardiac potassium channel called hERG can impair channel function by decreasing cell-surface channel expression, resulting in cardiac electrical disturbances and even sudden cardiac death. Promotion of cell-surface expression of mutant hERG channels represents a strategy to rescue channel function. This work demonstrates that drugs such as remdesivir and lumacaftor can differently affect homomeric and heteromeric mutant hERG channels, which have biological and clinical implications.

Early Steps in C-Type Inactivation of the hERG Potassium Channel.

Fast C-type inactivation confers distinctive functional properties to the hERG potassium channel, and its association to inherited and acquired cardiac arrythmias makes the study of the inactivation mechanism of hERG at the atomic detail of paramount importance. At present, two models have been proposed to describe C-type inactivation in K+-channels. Experimental data and computational work on the bacterial KcsA channel support the hypothesis that C-type inactivation results from a closure of the selectivity filter that sterically impedes ion conduction. Alternatively, recent experimental structures of a mutated Shaker channel revealed a widening of the extracellular portion of the selectivity filter, which might diminish conductance by interfering with the mechanism of ion permeation. Here, we performed molecular dynamics simulations of the wild-type hERG, a non-inactivating mutant (hERG-N629D), and a mutant that inactivates faster than the wild-type channel (hERG-F627Y) to find out which and if any of the two reported C-type inactivation mechanisms applies to hERG. Closure events of the selectivity filter were not observed in any of the simulated trajectories but instead, the extracellular section of the selectivity filter deviated from the canonical conductive structure of potassium channels. The degree of widening of the potassium binding sites at the extracellular entrance of the channel was directly related to the degree of inactivation with hERG-F627Y > wild-type hERG > hERG-N629D. These findings support the hypothesis that C-type inactivation in hERG entails a widening of the extracellular entrance of the channel rather than a closure of the selectivity filter.

Multiple mechanisms underlie reduced potassium conductance in the p.T1019PfsX38 variant of hERG.

Long QT syndrome type II (LQT2) is caused by loss‐of‐function mutations in the hERG K+ channel, leading to increased incidence of cardiac arrest and sudden death. Many genetic variants have been reported in the hERG gene with various consequences on channel expression, permeation, and gating. Only a small number of LQT2 causing variants has been characterized to define the underlying pathophysiological causes of the disease. We sought to determine the characteristics of the frameshift variant p.Thr1019ProfsX38 (T1019PfsX38) which affects the C‐terminus of the protein. This mutation was identified in an extended Omani family of LQT2. It replaces the last 140 amino acids of hERG with 37 unique amino acids. T1019 is positioned at a distinguished region of the C‐terminal tail of hERG, as predicted from the deep learning system AlphaFold v2.0. We employed the whole‐cell configuration of the patch‐clamp technique to study wild‐type and mutant channels that were transiently expressed in human embryonic kidney 293 (HEK293) cells. Depolarizing voltages elicited slowly deactivating tail currents that appeared upon repolarization of cells that express either wild‐type‐ or T1019PfsX38‐hERG. There were no differences in the voltage and time dependencies of activation between the two variants. However, the rates of hERG channel deactivation at hyperpolarizing potentials were accelerated by T1019PfsX38. In addition, the voltage dependence of inactivation of T1019PfsX38‐hERG was shifted by 20 mV in the negative direction when compared with wild‐type hERG. The rates of channel inactivation were increased in the mutant channel variant. Next, we employed a step‐ramp protocol to mimic membrane repolarization by the cardiac action potential. The amplitudes of outward currents and their integrals were reduced in the mutant variant when compared with the wild‐type variant during repolarization. Thus, changes in the gating dynamics of hERG by the T1019PfsX38 variant contribute to the pathology seen in affected LQT2 patients.

Mechanisms of gefitinib-induced QT prolongation

Gefitinib, a tyrosine kinase inhibitor, was the first targeted therapy for non-small cell lung cancer (NSCLC). Gefitinib could block human Ether-à-go-go-Related Gene (hERG) channel, an important target in drug-induced long QT syndrome. However, it is unclear whether gefitinib could induce QT interval prolongation. Here, whole-cell patch-clamp technique was used for evaluating the effect of gefitinib on rapidly-activating delayed rectifier K+ current (IKr), slowly-activating delayed rectifier K+ current (IKs), transient outward potassium current (Ito), inward rectifier K+ current (IK1) and on action potentials in guinea pig ventricular myocytes. The Langendorff heart perfusion technique was used to determine drug effect on the ECG. Gefitinib depressed IKr by binding to open and closed hERG channels in a concentration-dependent way (IC50: 1.91 μM). The inhibitory effect of gefitinib on wildtype hERG channels was reduced at the hERG mutants Y652A, S636A, F656V and S631A (IC50: 8.51, 13.97, 18.86, 32.99 μM), indicating that gefitinib is a pore inhibitor of hERG channels. In addition, gefitinib accelerated hERG channel inactivation and decreased channel steady-state inactivation. Gefitinib also decreased IKs with IC50 of 23.8 μM. Moreover, gefitinib increased action potential duration (APD) in guinea pig ventricular myocytes and the corrected QT interval (QTc) in isolated perfused guinea pig hearts in a concentration-dependent way (1–30 μM). These findings indicate that gefitinib could prolong QTc interval by potently blocking hERG channel, modulating kinetic properties of hERG channel. Partial block of KCNQ1/KCNE1 could also contribute to delayed repolarization and prolonged QT interval. Thus, caution should be taken when gefitinib is used for NSCLC treatment.

Serine mutation of a conserved threonine in the hERG K+ channel S6-pore region leads to loss-of-function through trafficking impairment

The human Ether-à-go-go Related Gene (hERG) encodes a potassium channel responsible for the cardiac rapid delayed rectifier K+ current, IKr, which regulates ventricular repolarization. Loss-of-function hERG mutations underpin the LQT2 form of congenital long QT syndrome. This study was undertaken to elucidate the functional consequences of a variant of uncertain significance, T634S, located at a highly conserved position at the top of the S6 helix of the hERG channel. Whole-cell patch-clamp recordings were made at 37 °C of hERG current (IhERG) from HEK 293 cells expressing wild-type (WT) hERG, WT+T634S and hERG-T634S alone. When the T634S mutation was expressed alone little or no IhERG could be recorded. Co-expressing WT and hERG-T634S suppressed IhERG tails by ∼57% compared to WT alone, without significant alteration of voltage dependent activation of IhERG. A similar suppression of IhERG was observed under action potential voltage clamp. Comparable reduction of IKr in a ventricular AP model delayed repolarization and led to action potential prolongation. A LI-COR® based On/In-Cell Western assay showed that cell surface expression of hERG channels in HEK 293 cells was markedly reduced by the T634S mutation, whilst total cellular hERG expression was unaffected, demonstrating impaired trafficking of the hERG-T634S mutant. Incubation with E−4031, but not lumacaftor, rescued defective hERG-T634S channel trafficking and IhERG density. In conclusion, these data identify hERG-T634S as a rescuable trafficking defective mutation that reduces IKr sufficiently to delay repolarization and, thereby, potentially produce a LQT2 phenotype.

Inhibition of hERG Potassium Channels by the Polycylic Aromatic Hydrocarbon Phenanthrene

Polycyclic aromatic hydrocarbons (PAHs) may contribute to cardiotoxic effects of hydrocarbon-based pollution. Phenanthrene (Phen) is an abundant PAH in polluted air and water. For example, Phen is a constituent PAH of crude oil spills and has been shown to increase ventricular action potential duration of fish ventricular myocytes. This action is associated with inhibition of the rapid delayed rectifier K+-current. Ether-à-go-go Related Gene encoded potassium channels (ERG) mediate IKr in multiple species including fish and human, and block of human ERG (hERG) channels by structurally diverse drugs can result in acquired long QT syndrome in humans. The purpose of this study was to investigate the action of Phen on hERG, using whole-cell patch-clamp recording from HEK 293 cells expressing wild-type (WT) or mutant hERG channels. Under conventional voltage-clamp, WT hERG current (IhERG) tails at −40 mV following an activating protocol were inhibited with an IC50 of 16.8 ± 1.7 µM, Hill slope: 1.0 ± 0.1 (mean ± SEM; n= at least 5 for 5 different concentrations). Phen (10 µM) inhibition of IhERG was voltage-dependent and was associated with leftward shift of voltage-dependent activation (V0.5 shift of −10.1 ± 0.9 mV; n=16). The extent of observed IhERG inhibition also varied with duration of the activating voltage command. IhERG carried by the attenuated inactivation N588K hERG mutant was markedly less sensitive to Phen and the F557L mutation in the S5 domain also reduced Phen inhibition. Thus, the observed IKr block with Phen appears to involve a direct inhibitory effect of the compound on (h)ERG channels that is contingent on channel gating. Intact inactivation gating and availability of the F557 residue appear to be significant influences on this inhibitory action. Funded by the British Heart Foundation PG/17/77/33125

The molecular determinants of R-roscovitine block of hERG channels.

Human ether-à-go-go-related gene (Kv11.1, or hERG) is a potassium channel that conducts the delayed rectifier potassium current (IKr) during the repolarization phase of cardiac action potentials. hERG channels have a larger pore than other K+channels and can trap many unintended drugs, often resulting in acquired LQTS (aLQTS). R-roscovitine is a cyclin-dependent kinase (CDK) inhibitor that induces apoptosis in colorectal, breast, prostate, multiple myeloma, other cancer cell lines, and tumor xenografts, in micromolar concentrations. It is well tolerated in phase II clinical trials. R-roscovitine inhibits open hERG channels but does not become trapped in the pore. Two-electrode voltage clamp recordings from Xenopus oocytes expressing wild-type (WT) or hERG pore mutant channels (T623A, S624A, Y652A, F656A) demonstrated that compared to WT hERG, T623A, Y652A, and F656A inhibition by 200 μM R-roscovitine was ~ 48%, 29%, and 73% weaker, respectively. In contrast, S624A hERG was inhibited more potently than WT hERG, with a ~ 34% stronger inhibition. These findings were further supported by the IC50 values, which were increased for T623A, Y652A and F656A (by ~5.5, 2.75, and 42 fold respectively) and reduced 1.3 fold for the S624A mutant. Our data suggest that while T623, Y652, and F656 are critical for R-roscovitine-mediated inhibition, S624 may not be. Docking studies further support our findings. Thus, R-roscovitine’s relatively unique features, coupled with its tolerance in clinical trials, could guide future drug screens.

Differential Regulation of Human Ether-à-Go-Go–Related Gene (hERG) Current and Expression by Activation of Protein Kinase C

The human ether-à-go-go-related gene (hERG) encodes the channel that conducts the rapidly activating delayed rectifier potassium current (IKr) in the heart. Reduction in IKr causes long QT syndrome, which can lead to fatal arrhythmias triggered by stress. One potential link between stress and hERG function is protein kinase C (PKC) activation; however, seemingly conflicting results regarding PKC regulation of hERG have been reported. We investigated the effects of PKC activation using phorbol 12-myristate 13-acetate (PMA) on hERG channels expressed in human embryonic kidney cell line 293 (HEK293) cells and IKr in isolated neonatal rat ventricular myocytes. Acute activation of PKC by PMA (30 nM, 30 minutes) reduced both hERG current (IhERG) and IKr Chronic activation of PKC by PMA (30 nM, 16 hours) increased IKr in cardiomyocytes and the expression level of hERG proteins; however, chronic (30 nM, 16 hours) PMA treatment decreased IhERG, which became larger than untreated control IhERG after PMA removal for 4 hours. Deletion of amino acid residues 2-354 (Δ2-354 hERG) or 1-136 of the N terminus (ΔN 136 hERG) abolished acute PMA (30 nM, 30 minutes)-mediated IhERG reduction. In contrast to wild-type hERG channels, chronic activation of PKC by PMA (30 nM, 16 hours) increased both Δ2-354 hERG and ΔN136 hERG expression levels and currents. The increase in hERG protein was associated with PKC-induced phosphorylation (inhibition) of Nedd4-2, an E3 ubiquitin ligase that mediates hERG degradation. We conclude that PKC regulates hERG in a balanced manner, increasing expression through inhibiting Nedd4-2 while decreasing current through targeting a site(s) within the N terminus.

An exploration of interactions between the antiarrhythmic drug dronedarone and hERG potassium channel pore

Dronedarone is a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone. It exerts potent inhibition of “hERG” potassium channels that underpin the cardiac rapid delayed rectifier potassium current, IKr. This study aimed to extend understanding of interactions between dronedarone and the hERG channel. Whole-cell patch-clamp recordings were made at 37?C of hERG channel current (IhERG) from HEK-293 cells expressing wild-type (WT) hERG or alanine mutants of residues in the channel’s pore-helix/selectivity filter region (T623, S624, V625) or S6 helices (S649, Y652, F656, V659). Molecular docking simulations were performed using a cryo-EM structure of hERG and a MthK-based homology model. The half-maximal inhibitory (IC50) value for WT IhERG inhibition by dronedarone was 42.6 ± 3.9 nM (n= at least 5 cells for each of 6 concentrations). 600 nM dronedarone exerted reduced WT IhERG block when the direction of K+ flux was reversed, consistent with interactions between the drug and permeant ion. In contrast with recently reported data for amiodarone, the S624A mutation did not attenuate IhERG blockade, whilst T623A and V625A channels exhibited modestly attenuated block. The S649A mutation was without significant effect and the Y652A and F656A mutations exhibited modest reductions in block. The V659A mutation produced the most marked effect on dronedarone action. Docking simulations were generally consistent with modest interactions with canonical binding residues and suggested an indirect rather than direct effect of the V659A mutation on the drug’s action. These findings leave open the possibility that as yet unexplored residue(s) could act as key determinants of high affinity hERG channel block by dronedarone.

Development of a High-Throughput Flow Cytometry Assay to Monitor Defective Trafficking and Rescue of Long QT2 Mutant hERG Channels.

Long QT Syndrome (LQTS) is an acquired or inherited disorder characterized by prolonged QT interval, exertion-triggered arrhythmias, and sudden cardiac death. One of the most prevalent hereditary LQTS subtypes, LQT2, results from loss-of-function mutations in the hERG channel, which conducts IKr, the rapid component of the delayed rectifier K+ current, critical for cardiac repolarization. The majority of LQT2 mutations result in Class 2 deficits characterized by impaired maturation and trafficking of hERG channels. Here, we have developed a high-throughput flow cytometric assay to analyze the surface and total expression of wild-type (WT) and mutant hERG channels with single-cell resolution. To test our method, we focused on 16 LQT2 mutations in the hERG Per-Arnt-Sim (PAS) domain that were previously studied via a widely used biochemical approach that compares levels of 135-kDa immature and 155-kDa fully glycosylated hERG protein to infer surface expression. We confirmed that LQT2 mutants expressed in HEK293 cells displayed a decreased surface density compared to WT hERG, and were differentially rescued by low temperature. However, we also uncovered some notable differences from the findings obtained via the biochemical approach. In particular, three mutations (N33T, R56Q, and A57P) with apparent WT-like hERG glycosylation patterns displayed up to 50% decreased surface expression. Furthermore, despite WT-like levels of complex glycosylation, these mutants have impaired forward trafficking, and exhibit varying half-lives at the cell surface. The results highlight utility of the surface labeling/flow cytometry approach to quantitatively assess trafficking deficiencies associated with LQT2 mutations, to discern underlying mechanisms, and to report on interventions that rescue deficits in hERG surface expression.

4. The Role of Glycosylation in Cardiac Arrhythmias

Long QT syndrome (LQTS) is a debilitating cardiac arrhythmia, and mutations or malfunctions in the human ether-a-go-go–related gene (hERG) are the most common cause of LQTS. hERG is responsible for the cardiac potassium current (IKr), which is important for cardiac repolarization. It has 6 transmembrane domains, ranging from S1 to S6, and a complex N-linked oligosaccharide chain resides on the extracellular S5 pore linker region. The role of this glycosylation chain in hERG function has not yet been well distinguished and in the current study we aim to investigate the relationship between hERG protease susceptibility and the presence of the glycosylation chain. Using molecular techniques, we demonstrate that de-glycosylated hERG channels are degraded by the serine protease Proteinase K at a much faster rate than their glycosylated counter parts. Additionally, through removal of the end-chain sialic acid residues using the enzyme neuraminidase, we conclude that it is likely the physical blockade of susceptible protease sites that grants wild type hERG this glycosylation-dependent protection as opposed to charge repulsion facilitated by specific sugars in the carbohydrate chain. Our data show that the glycosylation chain found on hERG plays a role in protecting the channel from protease degradation, and could provide valuable insight into the development of the cardiac arrhythmia Long QT Syndrome.

Ring Finger Protein 207 Degrades T613M Kv11.1 Channel

Introduction: Human ether-à-go-go related gene (hERG, Kv11.1) channel underlies cardiac rapidly activating delayed rectifier K+ current (IKr); hERG mutations contribute to Long QT Syndrome (LQTS). Additionally, reduced repolarization reserve can arise from not only ion channel mutations, but also mutations in ion channel interacting proteins. RING Finger Protein 207 (RNF207) has been identified as a potential modifier for Kv11.1 function, cardiac excitation, and QT interval. Recent studies suggested a link between hERG and RNF207 mutations in LQTS onset and severity, specifically T613M hERG (hERGT613M) and RNF207 frameshift mutation (RNF207G603fs). However, exact molecular mechanisms remain unknown. We hypothesize that RNF207G603fs fails to degrade hERGT613M, allowing a higher population of membrane-bound hERGT613M mutant subunits, thus decreasing functional IKr. Methods and Results: We confirmed ubiquitinylation activity in wild-type RNF207 (RNF207WT). Confocal and super-resolution microscopy in murine and rabbit ventricular cardiomyocytes confirmed co-localization of hERG with RNF207. Functional analyses revealed that the trafficking-defective hERGT613M failed to produce currents. Wild-type hERG (hERGWT) subunits only partially rescued hERGT613M with reduced IKr and faster activation and inactivation kinetics. RNF207WT co-expression with hERGWT and hERGT613M restored IKr, whereas RNF207G603fs failed to rescue IKr. Co-immunoprecipitation of hERGWT pulled down RNF207WT, but not RNF207G603fs. Further analysis using cytoplasmic N- and C-terminal hERGWT deletions (hERGWT-ΔN, hERGWT-ΔC) revealed that RNF207WT pulled down hERGWT-ΔN, but not hERGWT-ΔC. Additionally, time-dependent degradation assays demonstrated increased hERGT613M degradation by RNF207WT, but not RNF207G603fs, via proteasomal degradation. We further tested changes to RNF207 structure using Rosetta molecular modeling software. Conclusion: RNF207WT functions as an E3 ubiquitin ligase for hERG channels. RNF207G603fs, however, failed to degrade hERGT613M and rescue IKr density, suggesting that direct protein-protein interaction is necessary for observed rescue of current. Our data suggest that normal RNF207 function is critical for hERG quality control and, consequently, cardiac repolarization.

Intragenic Rescue of the Function of Long QT Syndrome-Causing hERG Mutant Channels

The human-ether-a-go-go-related gene (hERG) encodes the pore forming subunit of the rapidly activating delayed rectifier K+ current (IKr) which is important for the repolarization phase of cardiac action potentials. A reduction in IKr due to a loss of hERG function can lead to long QT syndrome (LQTS). There are a variety of loss-of-function hERG mutations known to cause LQTS, the majority of which are thought to be trafficking deficient, including G601S. It has been shown that the plasma membrane (PM) expression of the G601S hERG mutant can be rescued by reduced temperature culture. We have shown that wild-type (WT) hERG channels require extracellular K+ for their stability in the PM. They undergo internalization under 0 mM K+ culture conditions, which can be prevented by reduced temperature culture. We also identified that the S624T mutation makes hERG insensitive to extracellular K+, thereby preventing hERG channel internalization under 0 mM K+ culture conditions. We hypothesize that certain hERG mutants, including G601S, may not be able to sense extracellular K+, making them unstable in the PM, leading to a loss of PM expression. In this study, we added a secondary mutation, S624T, which does not depend on extracellular K+ for its PM expression, to the G601S mutant hERG channel. The addition of S624T suppressed the loss-of-function G601S phenotype, and rescued its current to a level similar to WT channels. The addition of S624T also rescued LQTS-causing hERG mutants T474I and P596R. Our data reveals the most effective intragenic rescue of LQTS-causing hERG mutants to date and lends insight into the mechanisms through which these mutations confer the loss-of-function phenotype.

Molecular mechanisms underlying the pilsicainide-induced stabilization of hERG proteins in transfected mammalian cells

BackgroundPilsicainide, classified as a relatively selective Na+ channel blocker, also has an inhibitory action on the rapidly-activating delayed-rectifier K+ current (IKr) through human ether-a-go-go-related gene (hERG) channels. We studied the effects of chronic exposure to pilsicainide on the expression of wild-type (WT) hERG proteins and WT-hERG channel currents, as well as on the expression of mutant hERG proteins, in a heterologous expression system. MethodsHEK293 cells stably expressing WT or mutant hERG proteins were subjected to Western blotting, immunofluorescence microscopy and patch-clamp experiments. ResultsAcute exposure to pilsicainide at 0.03–10μM influenced neither the expression of WT-hERG proteins nor WT-hERG channel currents. Chronic treatment with 0.03–10μM pilsicainide for 48h, however, increased the expression of WT-hERG proteins and channel currents in a concentration-dependent manner. Chronic treatment with 3μM pilsicainide for 48h delayed degradation of WT-hERG proteins and increased the channels expressed on the plasma membrane. A cell membrane-impermeant pilsicainide derivative did not influence the expression of WT-hERG, indicating that pilsicainide stabilized the protein inside the cell. Pilsicainide did not influence phosphorylation of Akt (protein kinase B) or expression of heat shock protein families such as HSF-1, hsp70 and hsp90. E4031, a chemical chaperone for hERG, abolished the pilsicainide effect on hERG. Chronic treatment with pilsicainide could also increase the protein expression of trafficking-defective mutant hERG, G601S and R752W. ConclusionsPilsicainide penetrates the plasma membrane, stabilizes WT-hERG proteins by acting as a chemical chaperone, and enhances WT-hERG channel currents. This mechanism could also be applicable to modulations of certain mutant-hERG proteins.

Propofol inhibits hERG K+ channels and enhances the inhibition effects on its mutations in HEK293 cells

QT interval prolongation, a potential risk for arrhythmias, may result from gene polymorphisms relevant to cardiomyocyte repolarization. Another noted cause of QT interval prolongation is the administration of chemical compounds such as anesthetics, which may affect a specific type of cardiac K+ channel encoded by the human ether-a-go-go-related gene (hERG).hERG K+ current was recorded using whole-cell patch clamp in human embryonic kidney (HEK293) cells expressing wild type (WT) or mutated hERG channels. Expression of hERG K+ channel proteins was evaluated using western blot and confirmed by fluorescent staining and imaging. Computational modeling was adopted to identify the possible binding site(s) of propofol with hERG K+ channels. Propofol had a significant inhibitory effect on WT hERG K+ currents in a concentration-dependent manner, with a half-maximal inhibitory concentration (IC50) of 60.9±6.4μM. Mutations in drug-binding sites (Y652A or F656C) of the hERG channel were found to attenuate hERG current blockage by propofol. However, propofol did not inhibit the trafficking of hERG protein to the cell membrane. Meanwhile, for the three selective hERG K+ channel mutant heterozygotes WT/Q738X-hERG, WT/A422T-hERG, and WT/H562P-hERG, the IC50 of propofol was calculated as 14.2±2.8μM, 3.3±1.2μM, and 5.9±1.9μM, respectively, which were much lower than that for the wild type.These findings indicate that propofol may potentially increase QT interval prolongation risk in patients via direct inhibition of the hERG K+ channel, especially in those with other concurrent triggering factors such as hERG gene mutations.

Histone Deacetylase Inhibitors Prolong Cardiac Repolarization through Transcriptional Mechanisms.

Histone deacetylase (HDAC) inhibitors are an emerging class of anticancer agents that modify gene expression by altering the acetylation status of lysine residues of histone proteins, thereby inducing transcription, cell cycle arrest, differentiation, and cell death or apoptosis of cancer cells. In the clinical setting, treatment with HDAC inhibitors has been associated with delayed cardiac repolarization and in rare instances a lethal ventricular tachyarrhythmia known as torsades de pointes. The mechanism(s) of HDAC inhibitor-induced effects on cardiac repolarization is unknown. We demonstrate that administration of structurally diverse HDAC inhibitors to dogs causes delayed but persistent increases in the heart rate corrected QT interval (QTc), an in vivo measure of cardiac repolarization, at timepoints far removed from the Tmax for parent drug and metabolites. Transcriptional profiling of ventricular myocardium from dogs treated with various HDAC inhibitors demonstrated effects on genes involved in protein trafficking, scaffolding and insertion of various ion channels into the cell membrane as well as genes for specific ion channel subunits involved in cardiac repolarization. Extensive in vitro ion channel profiling of various structural classes of HDAC inhibitors (and their major metabolites) by binding and acute patch clamp assays failed to show any consistent correlations with direct ion channel blockade. Drug-induced rescue of an intracellular trafficking-deficient mutant potassium ion channel, hERG (G601S), and decreased maturation (glycosylation) of wild-type hERG expressed by CHO cells in vitro correlated with prolongation of QTc intervals observed in vivo The results suggest that HDAC inhibitor-induced prolongation of cardiac repolarization may be mediated in part by transcriptional changes of genes required for ion channel trafficking and localization to the sarcolemma. These data have broad implications for the development of these drug classes and suggest that the optimal time to assess potentially transcriptionally mediated physiologic effects will be delayed relative to an epigenetic drug's Tmax/Cmax.

HERG 1a LQT2 C-terminus truncation mutants display hERG 1b-dependent dominant negative mechanisms

The human ether-à-go-go-related gene (hERG 1a) potassium channel is critical for cardiac repolarization. hERG 1b, another variant subunit, co-assembles with hERG 1a, modulates channel biophysical properties and plays an important role in repolarization. Mutations of hERG 1a lead to type 2 long QT syndrome (LQT2), and increased risk for fatal arrhythmias. The functional consequences of these mutations in the presence of hERG 1b are not known. To investigate whether hERG 1a mutants exert dominant negative gating and trafficking defects when co-expressed with hERG 1b. Electrophysiology, co-immunoprecipitation, and fluorescence resonance energy transfer (FRET) experiments in HEK293 cells and guinea pig cardiomyocytes were used to assess the mutants on gating and trafficking. Mutations of 1a-G965X and 1a-R1014X, relevant to gating and trafficking were introduced in the C-terminus region. The hERG 1a mutants when expressed alone did not result in decreased current amplitude. Compared to wild-type hERG 1a currents, 1a-G965X currents were significantly larger, whereas those produced by the 1a-R1014X mutant were similar in magnitude. Only when co-expressed with wild-type hERG 1a and 1b did a mutant phenotype emerge, with a marked reduction in surface expression, current amplitude, and a corresponding positive shift in the V1/2 of the activation curve. Co-immunoprecipitation and FRET assays confirmed association of mutant and wild-type subunits. Heterologously expressed hERG 1a C-terminus truncation mutants, exert a dominant negative gating and trafficking effect only when co-expressed with hERG 1b. These findings may have potentially profound implications for LQT2 therapy.

Characterization of the novel mutant A78T-HERG from a long QT syndrome type 2 patient: Instability of the mutant protein and stabilization by heat shock factor 1

BackgroundThe human ether-a-go-go-related gene (HERG) encodes the α-subunit of rapidly activating delayed-rectifier potassium channels. Mutations in this gene cause long QT syndrome type 2 (LQT2). In most cases, mutations reduce the stability of the channel protein, which can be restored by heat shock (HS). MethodsWe identified the novel mutant A78T-HERG in a patient with LQT2. The purpose of the current study was to characterize this mutant protein and test whether HS and heat shock factors (HSFs) could stabilize the mutant protein. A78T-HERG and wild-type HERG (WT-HERG) were expressed in HEK293 cells and analyzed by immunoblotting, immunoprecipitation, immunofluorescence, and whole-cell patch clamping. ResultsWhen expressed in HEK293 cells, WT-HERG gave rise to immature and mature forms of the protein at 135 and 155kDa, respectively. A78T-HERG gave rise only to the immature form, which was heavily ubiquitinated. The proteasome inhibitor MG132 increased the expression of immature A78T-HERG and increased both the immature and mature forms of WT-HERG. WT-HERG, but not A78T-HERG, was expressed on the plasma membrane. In whole-cell patch clamping experiments, depolarizing pulses evoked E4031-sensitive HERG channel currents in cells transfected with WT-HERG, but not in cells transfected with A78T-HERG. The A78V mutant, but not A78G mutant, remained in the immature form similarly to A78T. Maturation of the A78T-HERG protein was facilitated by HS, expression of HSF-1, or exposure to geranyl geranyl acetone. ConclusionsA78T-HERG was characterized by protein instability and reduced expression on the plasma membrane. The stability of the mutant was partially restored by HSF-1, indicating that HSF-1 is a target for the treatment for LQT2 caused by the A78T mutation in HERG.