Function Of hERG Research Articles

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

Investigation of the Inactivation of HERG using Molecular Dynamics Simulations of New cryo-EM Structures

The human ether á-go-go related gene (hERG) encodes the delayed rectifier potassium channel (Kv11.1), best known for mediation of the repolarising current in the cardiac action potential. Loss of function of hERG can lead to long QT-syndrome which causes cardiac arrhythmias and sudden cardiac arrest. hERG is susceptible to a wide range of drugs which can lead to drug induced long QT-syndrome. Here, the recent cryo-EM structure of hERG in an activated state, together with newly-solved structures that are open or inactivated serve as the basis to investigate the structural determinants of hERG inactivation. We used molecular dynamics flexible fitting to refine and relax these new structures in micelles consisting of detergents, lipids and cholesterol. A combination of long simulations and libraries of short simulations were performed with a variety of ion concentrations to study different conformational states. These simulations highlighted the amino acids important for maintaining each state of the selectivity filter and therefore may be involved in hERG inactivation. Furthermore, we have used replica exchange with solute tempering to enhance sampling of the exchange of ions in the hERG selectivity filter. We observe correlations between channel conformation and ion occupancy that shed light on the coupling of ions to inactivation. Together with mutagenesis experiments, these simulations provide molecular-level understanding of hERG inactivation, with considerable clinical and pharmacological potential.

Studying Structural and Dynamic Properties of KCNE3 in Various Membrane Environments Using Molecular Dynamics Simulation and EPR Spectroscopy

KCNE3 is a single transmembrane protein that modulates the function and trafficking of several voltage-gated potassium channels, including KCNQ1, KCNQ4, hERG, Kv2.1, Kv3.1, and Kv3.2. KCNE3 binds with KCNQ1 and plays a crucial role in potassium ion recycling, which is important for transepithelial chloride ion secretion. Mutations in KCNE3 are linked to diseases associated with salt and fluid disorders including cystic fibrosis (CF). Despite the biological significance, structural and dynamic properties of KCNE3 is not fully understood in different membrane environment. In this study, we investigate structural and dynamic properties of KCNE3 in different membrane environments such as POPC, POPC/POPG, DMPC, DOPC, using molecular dynamics simulation. The amino acid locations and side chain dynamics of WT KCNE3 will be compared to the mutated form of KCNE3. The molecular dynamics simulation studies will be compared with the continuous wave electron paramagnetic resonance (CW-EPR) lineshape analysis results.

Muscarinic Receptor Activation Increases hERG Channel Expression through Phosphorylation of Ubiquitin Ligase Nedd4-2

The human ether-à-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel, which is important for cardiac repolarization. Reduction of hERG current due to genetic mutations or drug interferences causes long QT syndrome, leading to cardiac arrhythmias and sudden death. To date, there is no effective therapeutic method to restore or enhance hERG channel function. Using cell biology and electrophysiological methods, we found that the muscarinic receptor agonist carbachol increased the expression and function of hERG, but not ether-à-go-go or Kv1.5 channels stably expressed in human embryonic kidney cells. The carbachol-mediated increase in hERG expression was abolished by the selective M3 antagonist 4-DAMP (1,1-dimethyl-4-diphenylacetoxypiperidinium iodide) but not by the M2 antagonist AF-DX 116 (11[[2-[(diethylamino)methyl]-1-piperidinyl]-acetyl]-5,11-dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepine-6-one). Treatment of cells with carbachol reduced the hERG-ubiquitin interaction and slowed the rate of hERG degradation. We previously showed that the E3 ubiquitin ligase Nedd4-2 mediates degradation of hERG channels. Here, we found that disrupting the Nedd4-2 binding domain in hERG completely eliminated the effect of carbachol on hERG channels. Carbachol treatment enhanced the phosphorylation level, but not the total level, of Nedd4-2. Blockade of the protein kinase C (PKC) pathway abolished the carbachol-induced enhancement of hERG channels. Our data suggest that muscarinic activation increases hERG channel expression by phosphorylating Nedd4-2 via the PKC pathway.

LQT2 nonsense mutations generate trafficking defective NH2-terminally truncated channels by the reinitiation of translation

The human ether-a-go-go-related gene (hERG) encodes a voltage-activated K(+) channel that contributes to the repolarization of the cardiac action potential. Long QT syndrome type 2 (LQT2) is an autosomal dominant disorder caused by mutations in hERG, and patients with LQT2 are susceptible to severe ventricular arrhythmias. We have previously shown that nonsense and frameshift LQT2 mutations caused a decrease in mutant mRNA by the nonsense-mediated mRNA decay (NMD) pathway. The Q81X nonsense mutation was recently found to be resistant to NMD. Translation of Q81X is reinitiated at Met(124), resulting in the generation of NH2-terminally truncated hERG channels with altered gating properties. In the present study, we identified two additional NMD-resistant LQT2 nonsense mutations, C39X and C44X, in which translation is reinitiated at Met(60). Deletion of the first 59 residues of the channel truncated nearly one-third of the highly structured Per-Arnt-Sim domain and resulted in the generation of trafficking-defective proteins and a complete loss of hERG current. Partial deletion of the Per-Arnt-Sim domain also resulted in the accelerated degradation of the mutant channel proteins. The coexpression of mutant and wild-type channels did not significantly disrupt the function and trafficking properties of wild-type hERG. Our present findings indicate that translation reinitiation may generate trafficking-defective as well as dysfunctional channels in patients with LQT2 premature termination codon mutations that occur early in the coding sequence.

An NMR investigation of the structure, function and role of the hERG channel selectivity filter in the long QT syndrome

The human ether-a-go-go-related gene (hERG) voltage-gated K+ channels are located in heart cell membranes and hold a unique selectivity filter (SF) amino acid sequence (SVGFG) as compared to other K+ channels (TVGYG). The hERG provokes the acquired long QT syndrome (ALQTS) when blocked, as a side effect of drugs, leading to arrhythmia or heart failure. Its pore domain – including the SF – is believed to be a cardiotoxic drug target. In this study combining solution and solid-state NMR experiments we examine the structure and function of hERG's L622-K638 segment which comprises the SF, as well as its role in the ALQTS using reported active drugs. We first show that the SF segment is unstructured in solution with and without K+ ions in its surroundings, consistent with the expected flexibility required for the change between the different channel conductive states predicted by computational studies. We also show that the SF segment has the potential to perturb the membrane, but that the presence of K+ ions cancels this interaction. The SF moiety appears to be a possible target for promethazine in the ALQTS mechanism, but not as much for bepridil, cetirizine, diphenhydramine and fluvoxamine. The membrane affinity of the SF is also affected by the presence of drugs which also perturb model DMPC-based membranes. These results thus suggest that the membrane could play a role in the ALQTS by promoting the access to transmembrane or intracellular targets on the hERG channel, or perturbing the lipid–protein synergy.

Effect of Daurisoline on hERG Channel Electrophysiological Function and Protein Expression

Daurisoline (1) is a bis-benzylisoquinoline alkaloid isolated from the rhizomes of Menispermum dauricum. The antiarrhythmic effect of 1 has been demonstrated in different experimental animals. In previous studies, daurisoline (1) prolonged action potential duration (APD) in a normal use-dependent manner. However, the electrophysiological mechanisms for 1-induced prolongation of APD have not been documented. In the present study, the direct effect of 1 was investigated on the hERG current and the expression of mRNA and protein in human embryonic kidney 293 (HEK293) cells stably expressing the hERG channel. It was shown that 1 inhibits hERG current in a concentration- and voltage-dependent manner. In the presence of 10 μM 1, steady-state inactivation of V(1/2) was shifted negatively by 15.9 mV, and 1 accelerated the onset of inactivation. Blockade of hERG channels was dependent on channel opening. The expression and function of hERG were unchanged by 1 at 1 and 10 μM, while hERG expression and the hERG current were decreased significantly by 1 at 30 μM. These results indicate that 1, at concentrations below 30 μM, exerts a blocking effect on hERG, but does not affect the expression and function of the hERG channel. This may explain the relatively lower risk of long QT syndrome after long-term usage.

Insights into hERG K+ channel structure and function from NMR studies

The unique gating kinetics of hERG K(+) channels are critical for normal cardiac repolarization, and patients with mutations in hERG have a markedly increased risk of cardiac arrhythmias and sudden cardiac arrest. HERG K(+) channels are also remarkably promiscuous with respect to drug binding, which has been a very significant problem for the pharmaceutical industry. Here, we review the progress that has been made in understanding the structure and function of hERG K(+) channels with a particular focus on nuclear magnetic resonance studies of the domains of the hERG K(+) channel.

Effect of the R1047L AF/LQT Mutation on the Distal C-Terminal Domain of the hERG Potassium Channel

Kv11.1 (hERG, KCNH2) is a voltage-gated potassium ion channel subunit involved in repolarization of cardiac action potential by mediating IKr current. Loss-of-function mutation or blockade of the channel pore by drug can induce a potentially fatal disorder called long QT syndrome (LQT). Atrial fibrillation (AF), the most commonly treated arrhythmia, has been linked to ion channel dysfunction. A significant proportion of AF patients have a positive family history and may therefore be affected by inherited mutations. We have studied gene variations in the C-terminal domain (CTD) of hERG isolated from patients with AF. One such variant, R1047L, is known to be associated with both AF and LQT, but its effects on the structure, stability, and function of hERG remain to be studied. Previous studies have shown that this site is within a coiled-coil domain that is thought to promote subunit assembly and form protein-protein interfaces. Electrophysiological studies showed difference in the tail current profile for wild-type (WT), R1047L, and the R1047L-WT heteromer. For structural studies, we made constructs of wt and R1047L mutant distal CTDs and purified with isotope labeling for high resolution NMR spectroscopy. 1H-15N HSQC spectrum of the WT distal CTD showed that the purified domain is consistent with predictions based on sequence analysis. Circular dichroism (CD) spectroscopy showed this domain is largely helical. Size-exclusion chromatography (SEC) and multi-angle light scattering (MALS) confirmed its molecular weight corresponds to a tetramer, as is expected for coiled-coil domains in voltage-gated ion channels. A comparison is presented of the stability and structural properties of the WT and R1047L mutant forms of this domain.

Novel Effects of Extracts from Poisonous Mushrooms on Expression and Function of the Human ether-a-go-go-Related Gene Channel

The human ether-a-go-go-related gene (hERG) encodes the α subunit of the potassium current I(Kr), which plays a pivotal role in cardiac action potential repolarization. Inherited mutations of this gene cause Long QT syndrome type 2. hERG expression is altered by several types of drugs as well as by temperature. Heat shock protein 70 (Hsp70) and Heat shock cognate protein 70 (Hsc70) have reciprocal effects on hERG proteins. We examined the effects of poisonous mushrooms on hERG protein expression and its channel function. We evaluated the effects of several types of poisonous mushrooms on the expression and function of wild-type hERG by Western blotting, reverse transcription polymerase chain reaction (PCR), and patch clamping in transfected HEK293 cells and mouse HL-1 cardiomyocytes. Extracts of Gymnopilus junonius (junonius) increased expression of both hERG and Hsp70 in HEK293 cells with concomitant decrease in Hsc70, whereas extracts of Amanita ibotengutake (ibotengutake) decreased hERG proteins with increase in Hsc70. Knockdown of Hsp70 and Hsc70 by small interfering RNA abolished the effects of the two mushrooms on hERG, respectively. Certain fractions of junonius increased expression of hERG proteins. hERG currents were increased by extracts of junonius, resulting in shortening of action potential duration (APD). In contrast, hERG currents were decreased and APD was prolonged by extracts of ibotengutake. junonius enhanced the expression and function of hERG by increasing Hsp70 and decreasing Hsc70. Ibotengutake decreased hERG expression via increase in Hsc70. Constituents of junonius may have the potential for use in treatment of arrhythmia.

Adrenergic Regulation of the Human Ether-A-Go-Go-Related Gene Channel Protein Abundance Occurs at the Surface of the Endoplasmic Reticulum

Long QT syndrome (LQTS) is a disorder of ion channels that can lead to the potentially lethal ventricular arrhythmias. Human ether-a-go-go-related gene product (HERG), a potassium channel responsible for the rapidly inactivating delayed rectifier current, is a genetic target in hereditary LQTS (specifically, LQTS2). Furthermore, pharmacological compounds of multiple classes can result in abnormal synthesis and/or function of HERG. Because arrhythmic events are often triggered in patients with LQTS2 following various stressors, it has been postulated that adrenergic regulation plays a key role in acute HERG regulation and LQTS pathogenesis. Recently, we have shown that chronic cAMP treatment (a surrogate for β-adrenergic signaling) significantly augments HERG protein abundance in HEK293 cells. Here, using velocity gradient centrifugations, we show that upon elevation of cAMP, the channel accumulates in the endoplasmic reticulum (ER) disproportionately more than at the plasma membrane. We also show that localized inhibition of protein kinase A (PKA) signaling at the surface of the ER by a targeted PKA inhibitory peptide (PKI) completely abolishes the effect; channels on the plasma membrane are unaffected by this inhibitor, as shown by patch-clamp experiments. Next, we targeted specific cAMP and PKA activity FRET-based biosensors (ICUE2 and AKAR3, respectively) to the ER surface; cell-permeable cAMP as well as the β-agonist isoproterenol elicited major FRET signal in the case of either biosensor, indicating that χAMΠ/ΠKA σιγναλινγ ισ ινταχτ ιν τηισ χoμπαρτμeντ. Tηe χAMΠ-δeπeνδeντ αυγμeντατιoν ρeμαινσ πρeσeντ eϖeν αϕτeρ τηe HEPΓ χoδινγ σeνeνχe ισ χoδoν-oπτιμιζeδ (38% διϕϕeρeντ ϕρoμ τηe oριγιναλ ατ τηe πριμαρψ σeνeνχe λeϖeλ), ινδιχατινγ τηατ χAMΠ-δeπeνδeντ αυγμeντατιoν ισ νoτ μPNA δeπeνδeντ. Tηeρeϕoρe, ωe χoνχλυδe τηατ β-adrenergic regulation of HERG occurs during the early stages of protein synthesis or folding at the level of the ER.

Regulation of HERG (KCNH2) potassium channel surface expression by diacylglycerol.

The HERG (KCNH2) channel is a voltage-sensitive potassium channel mainly expressed in cardiac tissue, but has also been identified in other tissues like neuronal and smooth muscle tissue, and in various tumours and tumour cell lines. The function of HERG has been extensively studied, but it is still not clear what mechanisms regulate the surface expression of the channel. In the present report, using human embryonic kidney cells stably expressing HERG, we show that diacylglycerol potently inhibits the HERG current. This is mediated by a protein kinase C-evoked endocytosis of the channel protein, and is dependent on the dynein-dynamin complex. The HERG protein was found to be located only in early endosomes and not lysosomes. Thus, diacylglycerol is an important lipid participating in the regulation of HERG surface expression and function.

Pharmacological separation of hEAG and hERG K+ channel function in the human mammary carcinoma cell line MCF-7

Pharmacological inhibitors of the human ether-a-go-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-a-go-go-related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [3H]-thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERG-specific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.

Combined hERG channel inhibition and disruption of trafficking in drug‐induced long QT syndrome by fluoxetine: a case‐study in cardiac safety pharmacology

Drug-induced prolongation of the rate-corrected QT interval (QTCI) on the electrocardiogram occurs as an unwanted effect of diverse clinical and investigational drugs and carries a risk of potentially fatal cardiac arrhythmias. hERG (human ether-à-go-go-related gene) is the gene encoding the alpha-subunit of channels mediating the rapid delayed rectifier K+ current, which plays a vital role in repolarising the ventricles of the heart. Most QTCI prolonging drugs can inhibit the function of recombinant hERG K+ channels, consequently in vitro hERG assays are used widely as front-line screens in cardiac safety-testing of novel chemical entities. In this issue, Rajamani and colleagues report a case of QTCI prolongation with the antidepressant fluoxetine and correlate this with a dual effect of the drug and of its major metabolite norfluoxetine on hERG channels. Both compounds were found to produce an acute inhibition of the hERG channel by pharmacological blockade, but in addition they also were able to disrupt the normal trafficking of hERG protein to the cell membrane. Mutations to a key component of the drug binding site in the S6 region of the channel greatly attenuated channel block, but did not impair disruption of trafficking; this suggests that channel block and drug effects on trafficking were mediated by different mechanisms. These findings add to growing evidence for disruption of hERG channel trafficking as a mechanism for drug-induced long QT syndrome and raise questions as to possible limitations of acute screening methods in the assessment of QTcI prolonging liability of drugs in development.

A mechanistic link between an inherited and an acquird cardiac arrthytmia: HERG encodes the I Kr potassium channel

Mutations in HERG cause an inherited cardiac arrhythmia, long OT syndrome (LOT). To define the function of HERG, we expressed the protein in Xenopus oocytes. The biophysical properties of expressed HERG are nearly identical to the rapidly activating delayed rectifier K + current (I Kr) in cardiac myocytes. HERG current is K + selective, declines with depolarizations above 0 mV, is activated by extracellular K +, and is blocked by lanthanum. Interestingly, HERG current is not blocked by drugs that specifically block I Kr in cardiac myocytes. These data indicate that HERG proteins form I Kr channels but that an additional subunit may be required for drug sensitivity. Since block of IKr is a known mechanism for drug-induced cardiac arrhythmias, the finding that HERG encodes IKr channels provides a mechanistic link between certain forms of inherited and acquired LOT.