Circadian rhythms govern cardiac repolarization and arrhythmogenesis.

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease, but the molecular basis of this variation is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (for example, short or long QT syndromes and heart failure) or pattern (for example, Brugada's syndrome) of myocardial repolarization. Here we provide molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion-channel expression and QT-interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a clock-dependent oscillator, krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of Kv channel-interacting protein 2 (KChIP2), a critical subunit required for generating the transient outward potassium current. Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. These findings identify circadian transcription of ion channels as a mechanism for cardiac arrhythmogenesis.

Ionic bases for electrical remodeling of the canine cardiac ventricle.

Emerging evidence suggests that ventricular electrical remodeling (VER) is triggered by regional myocardial strain via mechanoelectrical feedback mechanisms; however, the ionic mechanisms underlying strain-induced VER are poorly understood. To determine its ionic basis, VER induced by altered electrical activation in dogs undergoing left ventricular pacing (n = 6) were compared with unpaced controls (n = 4). Action potential (AP) durations (APDs), ionic currents, and Ca(2+) transients were measured from canine epicardial myocytes isolated from early-activated (low strain) and late-activated (high strain) left ventricular regions. VER in the early-activated region was characterized by minimal APD prolongation, but marked attenuation of the AP phase 1 notch attributed to reduced transient outward K(+) current. In contrast, VER in the late-activated region was characterized by significant APD prolongation. Despite marked APD prolongation, there was surprisingly minimal change in ion channel densities but a twofold increase in diastolic Ca(2+). Computer simulations demonstrated that changes in sarcolemmal ion channel density could only account for attenuation of the AP notch observed in the early-activated region but failed to account for APD remodeling in the late-activated region. Furthermore, these simulations identified that cytosolic Ca(2+) accounted for APD prolongation in the late-activated region by enhancing forward-mode Na(+)/Ca(2+) exchanger activity, corroborated by increased Na(+)/Ca(2+) exchanger protein expression. Finally, assessment of skinned fibers after VER identified altered myofilament Ca(2+) sensitivity in late-activated regions to be associated with increased diastolic levels of Ca(2+). In conclusion, we identified two distinct ionic mechanisms that underlie VER: 1) strain-independent changes in early-activated regions due to remodeling of sarcolemmal ion channels with no changes in Ca(2+) handling and 2) a novel and unexpected mechanism for strain-induced VER in late-activated regions in the canine arising from remodeling of sarcomeric Ca(2+) handling rather than sarcolemmal ion channels.

Molecular determinants of pentamidine-induced hERG trafficking inhibition.

Pentamidine is an antiprotozoal compound that clinically causes acquired long QT syndrome (acLQTS), which is associated with prolonged QT intervals, tachycardias, and sudden cardiac arrest. Pentamidine delays terminal repolarization in human heart by acutely blocking cardiac inward rectifier currents. At the same time, pentamidine reduces surface expression of the cardiac potassium channel I(Kr)/human ether à-go-go-related gene (hERG). This is unusual in that acLQTS is caused most often by direct block of the cardiac potassium current I(Kr)/hERG. The present study was designed to provide a more complete picture of how hERG surface expression is disrupted by pentamidine at the cellular and molecular levels. Using biochemical and electrophysiological methods, we found that pentamidine exclusively inhibits hERG export from the endoplasmic reticulum to the cell surface in a heterologous expression system as well as in cardiomyocytes. hERG trafficking inhibition could be rescued in the presence of the pharmacological chaperone astemizole. We used rescue experiments in combination with an extensive mutational analysis to locate an interaction site for pentamidine at phenylalanine 656, a crucial residue in the canonical drug binding site of terminally folded hERG. Our data suggest that pentamidine binding to a folding intermediate of hERG arrests channel maturation in a conformational state that cannot be exported from the endoplasmic reticulum. We propose that pentamidine is the founding member of a novel pharmacological entity whose members act as small molecule antichaperones.

Antidepressant-induced ubiquitination and degradation of the cardiac potassium channel hERG.

The most common cause for adverse cardiac events by antidepressants is acquired long QT syndrome (acLQTS), which produces electrocardiographic abnormalities that have been associated with syncope, torsade de pointes arrhythmias, and sudden cardiac death. acLQTS is often caused by direct block of the cardiac potassium current I(Kr)/hERG, which is crucial for terminal repolarization in human heart. Importantly, desipramine belongs to a group of tricyclic antidepressant compounds that can simultaneously block hERG and inhibit its surface expression. Although up to 40% of all hERG blockers exert combined hERG block and trafficking inhibition, few of these compounds have been fully characterized at the cellular level. Here, we have studied in detail how desipramine inhibits hERG surface expression. We find a previously unrecognized combination of two entirely different mechanisms; desipramine increases hERG endocytosis and degradation as a consequence of drug-induced channel ubiquitination and simultaneously inhibits hERG forward trafficking from the endoplasmic reticulum. This unique combination of cellular effects in conjunction with acute channel block may explain why tricyclic antidepressants as a compound class are notorious for their association with arrhythmias and sudden cardiac death. Taken together, we describe the first example of drug-induced channel ubiquitination and degradation. Our data are directly relevant to the cardiac safety of not only tricyclic antidepressants but also other therapeutic compounds that exert multiple effects on hERG, as hERG trafficking and degradation phenotypes may go undetected in most preclinical safety assays designed to screen for acLQTS.

Molecular coupling in the human ether-a-go-go-related gene-1 (hERG1) K+ channel inactivation pathway.

Emerging evidence suggests that K(+) channel inactivation involves coupling between residues in adjacent regions of the channel. Human ether-a-go-go-related gene-1 (hERG1) K(+) channels undergo a fast inactivation gating process that is crucial for maintaining electrical stability in the heart. The molecular mechanisms that drive inactivation in hERG1 channels are unknown. Using alanine scanning mutagenesis, we show that a pore helix residue (Thr-618) that points toward the S5 segment is critical for normal inactivation gating. Amino acid substitutions at position 618 modulate the free energy of inactivation gating, causing enhanced or reduced inactivation. Mutation of an S5 residue that is predicted to be adjacent to Thr-618 (W568L) abolishes inactivation and alters ion selectivity. The introduction of the Thr-618-equivalent residue in Kv1.5 enhances inactivation. Molecular dynamic simulations of the Kv1.2 tetramer reveal van der Waals coupling between hERG1 618- and 568-equivalent residues and a significant increase in interaction energies when threonine is introduced at the 618-equivalent position. We propose that coupling between the S5 segment and pore helix may participate in the inactivation process in hERG1 channels.

Oxidative inactivation of the lipid phosphatase phosphatase and tensin homolog on chromosome ten (PTEN) as a novel mechanism of acquired long QT syndrome.

The most common cause of cardiac side effects of pharmaco-therapy is acquired long QT syndrome, which is characterized by abnormal cardiac repolarization and most often caused by direct blockade of the cardiac potassium channel human ether a-go-go-related gene (hERG). However, little is known about therapeutic compounds that target ion channels other than hERG. We have discovered that arsenic trioxide (As(2)O(3)), a very potent antineoplastic compound for the treatment of acute promyelocytic leukemia, is proarrhythmic via two separate mechanisms: a well characterized inhibition of hERG/I(Kr) trafficking and a poorly understood increase of cardiac calcium currents. We have analyzed the latter mechanism in the present study using biochemical and electrophysiological methods. We find that oxidative inactivation of the lipid phosphatase PTEN by As(2)O(3) enhances cardiac calcium currents in the therapeutic concentration range via a PI3Kα-dependent increase in phosphatidylinositol 3,4,5-triphosphate (PIP(3)) production. In guinea pig ventricular myocytes, even a modest reduction in PTEN activity is sufficient to increase cellular PIP(3) levels. Under control conditions, PIP(3) levels are kept low by PTEN and do not affect calcium current amplitudes. Based on pharmacological experiments and intracellular infusion of PIP(3), we propose that in guinea pig ventricular myocytes, PIP(3) regulates calcium currents independently of the protein kinase Akt along a pathway that includes a secondary oxidation-sensitive target. Overall, our report describes a novel form of acquired long QT syndrome where the target modified by As(2)O(3) is an intracellular signaling cascade.

Phosphoinositide binding differentially regulates NHE1 Na+/H+ exchanger-dependent proximal tubule cell survival.

Tubular atrophy predicts chronic kidney disease progression, and is caused by proximal tubular epithelial cellcaused by proximal tubular epithelial cell (PTC) apoptosis. The normally quiescent Na(+)/H(+) exchanger-1 (NHE1) defends against PTC apoptosis, and is regulated by PI(4,5)P(2) binding. Because of the vast array of plasma membrane lipids, we hypothesized that NHE1-mediated cell survival is dynamically regulated by multiple anionic inner leaflet phospholipids. In membrane overlay and surface plasmon resonance assays, the NHE1 C terminus bound phospholipids with low affinity and according to valence (PIP(3) > PIP(2) > PIP = PA > PS). NHE1-phosphoinositide binding was enhanced by acidic pH, and abolished by NHE1 Arg/Lys to Ala mutations within two juxtamembrane domains, consistent with electrostatic interactions. PI(4,5)P(2)-incorporated vesicles were distributed to apical and lateral PTC domains, increased NHE1-regulated Na(+)/H(+) exchange, and blunted apoptosis, whereas NHE1 activity was decreased in cells enriched with PI(3,4,5)P(3), which localized to basolateral membranes. Divergent PI(4,5)P(2) and PI(3,4,5)P(3) effects on NHE1-dependent Na(+)/H(+) exchange and apoptosis were confirmed by selective phosphoinositide sequestration with pleckstrin homology domain-containing phospholipase Cδ and Akt peptides, PI 3-kinase, and Akt inhibition in wild-type and NHE1-null PTCs. The results reveal an on-off switch model, whereby NHE1 toggles between weak interactions with PI(4,5)P(2) and PI(3,4,5)P(3). In response to apoptotic stress, NHE1 is stimulated by PI(4,5)P(2), which leads to PI 3-kinase activation, and PI(4,5)P(2) phosphorylation. The resulting PI(3,4,5)P(3) dually stimulates sustained, downstream Akt survival signaling, and dampens NHE1 activity through competitive inhibition and depletion of PI(4,5)P(2).

Gap junction heterogeneity as mechanism for electrophysiologically distinct properties across the ventricular wall.

Gap junctions are critical to maintaining synchronized impulse propagation and repolarization. Heterogeneous expression of the principal ventricular gap junction protein connexin43 (Cx43) is associated with action potential duration (APD) dispersion across the anterior ventricular wall. Little is known about Cx43 expression patterns and their disparate impact on regional electrophysiology throughout the heart. We aimed to determine whether the anterior and posterior regions of the heart are electrophysiologically distinct. Multisegment, high-resolution optical mapping was performed in canine wedge preparations harvested separately from the anterior left ventricle (aLV; n = 8) and posterior left ventricle (pLV; n = 8). Transmural APD dispersion was significantly greater on the aLV than the pLV (45 +/- 13 vs. 26 +/- 8.0 ms; P < 0.05). Conduction velocity dispersion was also significantly higher (P < 0.05) across the aLV (39 +/- 7%) than the pLV (16 +/- 3%). Carbenoxolone perfusion significantly enhanced APD and conduction velocity dispersion on the aLV (by 1.53-fold and 1.36-fold, respectively), but not the pLV (by 1.27-fold and 1.2-fold, respectively), and produced a 4.2-fold increase in susceptibility to inducible arrhythmias in the aLV. Confocal immunofluorescence microscopy revealed significantly (P < 0.05) greater transmural dispersion of Cx43 expression on the aLV (44 +/- 10%) compared with the pLV wall (8.3 +/- 0.7%), suggesting that regional expression of Cx43 expression patterns may account for regional electrophysiological differences. Computer simulations affirmed that localized uncoupling at the epicardial-midmyocardial interface is sufficient to produce APD gradients observed on the aLV. These data demonstrate that the aLV and pLV differ importantly with respect to their electrophysiological properties and Cx43 expression patterns. Furthermore, local underexpression of Cx43 is closely associated with transmural electrophysiological heterogeneity on the aLV. Therefore, regional and transmural heterogeneous Cx43 expression patterns may be an important mechanism underlying arrhythmia susceptibility, particularly in disease states where gap junction expression is altered.

Intracellular potassium stabilizes human ether-à-go-go-related gene channels for export from endoplasmic reticulum.

Several therapeutic compounds have been identified that prolong the QT interval on the electrocardiogram and cause torsade de pointes arrhythmias not by direct block of the cardiac potassium channel human ether-à-go-go-related gene (hERG) but via disruption of hERG trafficking to the cell surface membrane. One example of a clinically important compound class that potently inhibits hERG trafficking are cardiac glycosides. We have shown previously that inhibition of hERG trafficking by cardiac glycosides is initiated via direct block of Na(+)/K(+) pumps and not via off-target interactions with hERG or any other protein. However, it was not known how pump inhibition at the cell surface is coupled to hERG processing in the endoplasmic reticulum. Here, we show that depletion of intracellular K(+)-either indirectly after long-term exposure to cardiac glycosides or directly after exposure to gramicidin in low sodium media-is sufficient to disrupt hERG trafficking. In K(+)-depleted cells, hERG trafficking can be restored by permeating K(+) or Rb(+) ions, incubation at low temperature, exposure to the pharmacological chaperone astemizole, or specific mutations in the selectivity filter of hERG. Our data suggest a novel mechanism for drug-induced trafficking inhibition in which cardiac glycosides produce a [K(+)](i)-mediated conformational defect directly in the hERG channel protein.

Hypoxia inhibits maturation and trafficking of hERG K(+) channel protein: Role of Hsp90 and ROS.

We previously reported that reactive oxygen species (ROS) generated during hypoxia decrease hERG current density and protein expression in HEK cells stably expressing hERG protein. In the present study, we investigated the molecular mechanisms involved in hypoxia-induced downregulation of hERG protein. Culturing cells at low temperatures and addition of chemical chaperones during hypoxia restored hERG expression and currents to normoxic levels while antiarrhythmic drugs, which selectively block hERG channels, had no effect on hERG protein levels. Pulse chase studies showed that hypoxia blocks maturation of the core glycosylated form in the endoplasmic reticulum (ER) to the fully glycosylated form on the cell surface. Co-immunoprecipitation experiments revealed that hypoxia inhibited interaction of hERG with Hsp90 chaperone required for maturation, which was restored in the presence of ROS scavengers. These results demonstrate that ROS generated during hypoxia prevents maturation of the hERG protein by inhibiting Hsp90 interaction resulting in decreased protein expression and currents.

Mitochondrial reactive oxygen species mediate hypoxic down-regulation of hERG channel protein.

Previous studies suggest that reactive oxygen species (ROS) play an important role in physiological responses to hypoxia. In the present study, we examined the effects of hypoxia on human ether-a-go-go related gene (hERG) channel protein expression and assessed the role of ROS. Hypoxia, in a stimulus- and time-dependent manner, decreased hERG protein with marked reduction in hERG K+ conductance in human embryonic kidney cells stably expressing the hERG alpha subunit. Down-regulation of hERG by hypoxia was not due to increased proteasomal degradation or decreased transcription but due to decreased synthesis of the protein. Hypoxia increased ROS in a time-dependent manner. Antioxidants prevented hypoxia-evoked down-regulation of hERG protein and exogenous oxidants mimicked the effects of hypoxia. Hypoxia-evoked down-regulation of hERG protein and elevation in ROS were absent in p(O) cells, which are devoid of mitochondrial DNA. Inhibitors of NADPH oxidase failed to prevent the effects of hypoxia. These results demonstrate that hypoxia enhances the production of ROS in the mitochondria, resulting in down-regulation of hERG translation and decreased hERG-mediated K+ conductance.

Doxazosin induces apoptosis of cells expressing hERG K+ channels.

The antihypertensive drug doxazosin has been associated with an increased risk for congestive heart failure and cardiomyocyte apoptosis. Human ether-a-go-go-related gene (hERG) K(+) channels, previously shown to be blocked by doxazosin at therapeutically relevant concentrations, represent plasma membrane receptors for the antihypertensive drug. To elucidate the molecular basis for doxazosin-associated pro-apoptotic effects, cell death was studied in human embryonic kidney cells using three independent apoptosis assays. Doxazosin specifically induced apoptosis in hERG-expressing HEK cells, while untransfected control groups were insensitive to treatment with the antihypertensive agent. An unexpected biological mechanism has emerged: binding of doxazosin to its novel membrane receptor, hERG, triggers apoptosis, possibly representing a broader pathophysiological mechanism in drug-induced heart failure.

Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity.

Congenital long QT syndrome (LQTS) is an inherited channelopathy associated with life-threatening arrhythmias. LQTS type 2 (LQT2) is caused by mutations in KCNH2, which encodes the potassium channel hERG. We hypothesized that modifier genes are partly responsible for the variable phenotype severity observed in some LQT2 families. Here, we identified contributors to variable expressivity in an LQT2 family by using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) and whole exome sequencing in a synergistic manner. We found that iPSC-CMs recapitulated the clinical genotype-phenotype discordance in vitro. Importantly, iPSC-CMs derived from the severely affected LQT2 patients displayed prolonged action potentials compared with cells from mildly affected first-degree relatives. The iPSC-CMs derived from all patients with hERG R752W mutation displayed lower IKr amplitude. Interestingly, iPSC-CMs from severely affected mutation-positive individuals exhibited greater L-type Ca2+ current. Whole exome sequencing identified variants of KCNK17 and the GTP-binding protein REM2, providing biologically plausible explanations for this variable expressivity. Genome editing to correct a REM2 variant reversed the enhanced L-type Ca2+ current and prolonged action potential observed in iPSC-CMs from severely affected individuals. Thus, our findings showcase the power of combining complementary physiological and genomic analyses to identify genetic modifiers and potential therapeutic targets of a monogenic disorder. Furthermore, we propose that this strategy can be deployed to unravel myriad confounding pathologies displaying variable expressivity.

KChIP2 is a core transcriptional regulator of cardiac excitability.

Arrhythmogenesis from aberrant electrical remodeling is a primary cause of death among patients with heart disease. Amongst a multitude of remodeling events, reduced expression of the ion channel subunit KChIP2 is consistently observed in numerous cardiac pathologies. However, it remains unknown if KChIP2 loss is merely a symptom or involved in disease development. Using rat and human derived cardiomyocytes, we identify a previously unobserved transcriptional capacity for cardiac KChIP2 critical in maintaining electrical stability. Through interaction with genetic elements, KChIP2 transcriptionally repressed the miRNAs miR-34b and miR-34c, which subsequently targeted key depolarizing (INa) and repolarizing (Ito) currents altered in cardiac disease. Genetically maintaining KChIP2 expression or inhibiting miR-34 under pathologic conditions restored channel function and moreover, prevented the incidence of reentrant arrhythmias. This identifies the KChIP2/miR-34 axis as a central regulator in developing electrical dysfunction and reveals miR-34 as a therapeutic target for treating arrhythmogenesis in heart disease.

Voltage-gated sodium channels assemble and gate as dimers.

Fast opening and closing of voltage-gated sodium channels are crucial for proper propagation of the action potential through excitable tissues. Unlike potassium channels, sodium channel α-subunits are believed to form functional monomers. Yet, an increasing body of literature shows inconsistency with the traditional idea of a single α-subunit functioning as a monomer. Here we demonstrate that sodium channel α-subunits not only physically interact with each other but they actually assemble, function and gate as a dimer. We identify the region involved in the dimerization and demonstrate that 14-3-3 protein mediates the coupled gating. Importantly we show conservation of this mechanism among mammalian sodium channels. Our study not only shifts conventional paradigms in regard to sodium channel assembly, structure, and function but importantly this discovery of the mechanism involved in channel dimerization and biophysical coupling could open the door to new approaches and targets to treat and/or prevent sodium channelopathies.

Role of the cytosolic chaperones Hsp70 and Hsp90 in maturation of the cardiac potassium channel HERG.

The human ether-a-gogo-related gene (hERG) encodes the alpha subunit of the cardiac potassium current IKr. Several mutations in hERG produce trafficking-deficient channels that may cause hereditary long-QT syndrome and sudden cardiac death. Although hERG currents have been studied extensively, little is known about the proteins involved in maturation and trafficking of hERG. Using immunoprecipitations, we show that the cytosolic chaperones heat shock protein (Hsp) 70 and Hsp90, but not Grp94, interact with hERG wild type (WT) during maturation. The specific Hsp90 inhibitor geldanamycin prevents maturation and increases proteasomal degradation of hERG WT, while reducing hERG currents in heterologous expression systems. In ventricular myocytes, inhibition of Hsp90 also decreases IKr, whereas geldanamycin had no effect on IKs or heterologously expressed Kv2.1 and Kv1.5 currents. Both Hsp90 and Hsp70 interact directly with the core-glycosylated form of hERG WT present in the endoplasmic reticulum but not the fully glycosylated, cell-surface form. For the trafficking-deficient LQT2 mutants, hERG R752W and hERG G601S, interactions with Hsp90 and Hsp70 are increased as both mutants remained tightly associated with Hsp90 and Hsp70 in the endoplasmic reticulum. Incubation at lower temperature for R752W or with the hERG blocker astemizole for G601S dissociates channel-chaperone complexes and restores trafficking. In contrast, nonfunctional but trafficking-competent hERG G628S is released from chaperone complexes during maturation comparable to WT. We conclude that Hsp90 and Hsp70 are crucial for the maturation of hERG WT as well as the retention of trafficking-deficient LQT2 mutants. The full text of this article is available online at http://www.circresaha.org.

HERG channel trafficking.

Mutations in the cardiac potassium channel hERG/IKr cause inherited long QT syndrome with increased susceptibility to ventricular arrhythmias. Several mutations in hERG produce trafficking-deficient channels that are retained in the endoplasmic reticulum (ER). Surface expression of certain mutations (i.e. hERG G601S) can be restored by specific channel blockers. Although hERG currents have been studied extensively, little is known about proteins in the processing pathway. Using biochemical and electrophysiological assays we show that the cytosolic chaperones Hsp70 and Hsp90 interact transiently with wild-type hERG. Inhibition of Hsp90 prevents maturation and reduces hERG/IKr currents. Trafficking-deficient mutants remain tightly associated with chaperones in the ER until trafficking is restored, e.g. by channel blockers. hERG/chaperone complexes represent novel targets for therapeutic compounds with cardiac liability such as arsenic, which is used in the treatment of leukaemias. Arsenic interferes with the formation of hERG/chaperone complexes and inhibits hERG maturation causing ECG abnormalities. We conclude that Hsp9O and Hsp70 are crucial for productive folding of wild-type hERG. Therapeutic compounds that inhibit chaperone function produce a novel form of acquired long QT syndrome not by direct channel block but by reduced surface expression due to an acquired trafficking defect of hERG.

HERG-Lite: a novel comprehensive high-throughput screen for drug-induced hERG risk.

INTRODUCTION: Direct block of I(Kr) by non-antiarrhythmic drugs (NARDs) is a major cause of QT prolongation and torsades de pointes (TdP), and has made the hERG potassium channel a major target of drug safety programs in cardiotoxicity. Block of hERG currents is not the only way that drugs can adversely impact the repolarizing current I(Kr), however. We have shown recently that two drugs in clinical use do not block hERG but produce long QT syndrome (LQTS) and TdP by inhibiting trafficking of hERG to the cell surface. To address the need for an inexpensive, rapid, and comprehensive assay to predict both types of hERG risk early in the drug development process, we have developed a novel antibody-based chemiluminescent assay called HERG-Lite. METHODS: HERG-Lite monitors the expression of hERG at the cell surface in two different stable mammalian cell lines. One cell line acts as a biosensor for drugs that inhibit hERG trafficking, while the other predicts hERG blockers based on their ability to act as pharmacological chaperones. In this study, we have validated the HERG-Lite assay using a panel of 100 drugs: 50 hERG blockers and 50 nonblockers. RESULTS: HERG-Lite correctly predicted hERG risk for all 100 test compounds with no false positives or negatives. All 50 hERG blockers were detected as drugs with hERG risk in the HERG-Lite assay, and fell into two classes: B (for blocker) and C (for complex; block and trafficking inhibition). DISCUSSION: HERG-Lite is the most comprehensive assay available for predicting drug-induced hERG risk. It accurately predicts both channel blockers and trafficking inhibitors in a rapid, cost-effective manner and is a valuable non-clinical assay for drug safety testing.

Direct block of hERG potassium channels by the protein kinase C inhibitor bisindolylmaleimide I (GF109203X).

OBJECTIVE: The human ether-a-go-go-related gene (hERG) encodes the rapid component of the cardiac repolarizing delayed rectifier potassium current, I(Kr). The direct interaction of the commonly used protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM I) with hERG, KvLQT1/minK, and I(Kr) currents was investigated in this study. METHODS: hERG and KvLQT1/minK channels were heterologously expressed in Xenopus laevis oocytes, and currents were measured using the two-microelectrode voltage clamp technique. In addition, hERG currents in stably transfected human embryonic kidney (HEK 293) cells, native I(Kr) currents and action potentials in isolated guinea pig ventricular cardiomyocytes were recorded using whole-cell patch clamp electrophysiology. RESULTS: Bisindolylmaleimide I blocked hERG currents in HEK 293 cells and Xenopus oocytes in a concentration-dependent manner with IC(50) values of 1.0 and 13.2 muM, respectively. hERG channels were primarily blocked in the open state in a frequency-independent manner. Analysis of the voltage-dependence of block revealed a reduction of inhibition at positive membrane potentials. BIM I caused a shift of -20.3 mV in the voltage-dependence of inactivation. The point mutations tyrosine 652 alanine (Y652A) and phenylalanine 656 alanine (F656A) attenuated hERG current blockade, indicating that BIM I binds to a common drug receptor within the pore region. KvLQT1/minK currents were not significantly altered by BIM I. Finally, 1 muM BIM I reduced native I(Kr) currents by 69.2% and lead to action potential prolongation. CONCLUSION: In summary, PKC-independent effects have to be carefully considered when using BIM I as PKC inhibitor in experimental models involving hERG channels and I(Kr) currents.

The antihistamine fexofenadine does not affect I(Kr) currents in a case report of drug-induced cardiac arrhythmia.

1. The human HERG gene encodes the cardiac repolarizing K(+) current I(Kr) and is genetically inactivated in inherited long QT syndrome 2 (LQTS2). The antihistamine terfenadine blocks HERG channels, and can cause QT prolongation and torsades de pointes, whereas its carboxylate fexofenadine lacks HERG blocking activity. 2. In the present study the ability of fexofenadine to block the K897T HERG channel variant was investigated. The underlying single nucleotide polymorphism (SNP) A2960C was identified in a patient reported to develop fexofenadine-associated LQTS. 3. K897T HERG channels produced wild-type-like currents in Xenopus oocytes. Even at a concentration of 100 micro M, fexofenadine did not inhibit wild-type or K897T HERG channels. Coexpression of wild-type and K897T HERG with the ss-subunit MiRP1, slightly changed current kinetics but did not change sensitivity to terfenadine and fexofenadine. 4. Western blot analysis and immunostaining of transiently transfected COS-7 cells demonstrated that overall expression level, glycosylation pattern and subcellular localization of K897T HERG is indistinguishable from wild-type HERG protein, and not altered in the presence of 1 micro M fexofenadine. 5. We provide the first functional characterization of the K897T HERG variant. We demonstrated that K897T HERG is similar to wild-type HERG, and is insensitive to fexofenadine. Although the polymorphism changes PKA and PKC phosphorylation sites, regulation of K897T HERG by these kinases is not altered. 6. Our results strongly indicate that QT lengthening and cardiac arrhythmia in the reported case of drug-induced LQT are not due to the K897T exchange or to an inhibitory effect of fexofenadine on cardiac I(Kr) currents. British Journal of