Mutant Human Ether-a-go-go-related Gene Research Articles

Progress in research on defective protein trafficking and functional restoration in HERG-associated long QT syndrome

The human ether-a-go-go related gene (HERG) encodes the α -subunit of the rapid component of the delayed rectifier K(+) channel, which is essential for the third repolarization of the action potential of human myocardial cells. Mutations of the HERG gene can cause type II hereditary long QT syndrome (LQT2), characterized by prolongation of the QT interval, abnormal T wave, torsade de pointes, syncope and sudden cardiac death. So far more than 300 HERG mutations have been identified, the majority of which can cause LQT2 due to HERG protein trafficking defect. It has been reported that certain drugs can induce acquired long QT syndrome through directly blocking the pore and/or affecting the HERG trafficking. The trafficking defects and K(+) currents can be restored with low temperature and certain drugs. However, the mechanisms underlying defective trafficking caused by HERG mutations and the inhibition/restoration of HERG trafficking by drugs are still unknown. This review summarizes the current understanding of the molecular mechanisms including HERG trafficking under physiological and pathological conditions, and the effects of drugs on the HERG trafficking, in order to provide theoretical evidence for the diagnosis and treatment of long QT syndrome.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

The evolutionarily conserved residue A653 plays a key role in HERG channel closing

Human ether-a-go-go-related gene (HERG) encodes the rapid, outwardly rectifying K(+) current I(Kr) that is critical for repolarization of the cardiac action potential. Congenital HERG mutations or unintended pharmaceutical block of I(Kr) can lead to life-threatening arrhythmias. Here, we assess the functional role of the alanine at position 653 (HERG-A653) that is highly conserved among evolutionarily divergent K(+) channels. HERG-A653 is close to the 'glycine hinge' implicated in K(+) channel opening, and is flanked by tyrosine 652 and phenylalanine 656, which contribute to the drug binding site. We substituted an array of seven (I, C, S, G, Y, V and T) amino acids at position 653 and expressed individual variants in heterologous systems to assess changes in gating and drug binding. Substitution of A653 resulted in negative shifts of the V(1/2) of activation ranging from -23.6 (A653S) to -62.5 (A653V) compared to -11.2 mV for wild-type (WT). Deactivation was also drastically altered: channels with A653I/C substitutions exhibited delayed deactivation in response to test potentials above the activation threshold, while A653S/G/Y/V/T failed to deactivate under those conditions and required hyperpolarization and prolonged holding potentials at -130 mV. While A653S/G/T/Y variants showed decreased sensitivity to the I(Kr) inhibitor dofetilide, these changes could not be correlated with defects in channel closure. Homology modelling suggests that in the closed state, A653 forms tight contacts with several residues from the neighbouring subunit in the tetramer, playing a key role in S6 helix packing at the narrowest part of the vestibule. Our study suggests that A653 plays an important functional role in the outwardly rectifying gating behaviour of HERG, supporting channel closure at membrane potentials negative to the channel activation threshold.

HERG biosynthesis: the positive influence of negative charge

HERG biosynthesis: the positive influence of negative charge

State-dependent block of HERG potassium channels by R-roscovitine: implications for cancer therapy

Human ether-a-go-go-related gene (HERG) potassium channel acts as a delayed rectifier in cardiac myocytes and is an important target for both pro- and antiarrhythmic drugs. Many drugs have been pulled from the market for unintended HERG block causing arrhythmias. Conversely, recent evidence has shown that HERG plays a role in cell proliferation and is overexpressed both in multiple tumor cell lines and in primary tumor cells, which makes HERG an attractive target for cancer treatment. Therefore, a drug that can block HERG but that does not induce cardiac arrhythmias would have great therapeutic potential. Roscovitine is a cyclin-dependent kinase (CDK) inhibitor that is in phase II clinical trials as an anticancer agent. In the present study we show that R-roscovitine blocks HERG potassium current (human embryonic kidney-293 cells stably expressing HERG) at clinically relevant concentrations. The block (IC(50) = 27 microM) was rapid (tau = 20 ms) and reversible (tau = 25 ms) and increased with channel activation, which supports an open channel mechanism. Kinetic study of wild-type and inactivation mutant HERG channels supported block of activated channels by roscovitine with relatively little effect on either closed or inactivated channels. A HERG gating model reproduced all roscovitine effects. Our model of open channel block by roscovitine may offer an explanation of the lack of arrhythmias in clinical trials using roscovitine, which suggests the utility of a dual CDK/HERG channel block as an adjuvant cancer therapy.

Inhibition of the HERG Channel by Droperidol Depends on Channel Gating and Involves the S6 Residue F656: Retracted

Droperidol has a central antiemetic action and is widely used in the fields of psychiatry, anesthesia, and emergency medicine. It has been associated with prolongation of the QT interval of the electrocardiogram, and it may also be associated with torsades de pointes and sudden death. Although QT prolongation is consistent with droperidol-induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been clearly studied. The rapidly activating delayed rectifier potassium channel, IKr, is a primary site of action of drugs causing QT prolongation and is encoded by the human-ether-a-go-go-related gene (HERG). To determine the mechanism underlying these clinical findings, we investigated the effect of droperidol on human HERG potassium channels. Wild type and mutant HERG channels were heterologously expressed in human embryonic kidney 293 cells, and the current was recorded by using whole cell patch clamp technique (22-24 degrees C). HERG tail currents following test pulses to 50 mV were inhibited by droperidol with an IC(50) of 77.3 +/- 9.6 nM (n = 8). The onset of block was fast and inhibition was completely reversible upon washout. Droperidol affected HERG channels mainly in their open and inactivated states. The effects were use-dependent with a stronger steady-state level of block at higher frequencies. The activation curve was slightly shifted towards more negative potentials (P < 0.05, n = 8) and the time course of inactivation was significantly decreased (P < 0.05, n = 8) by 100 nM droperidol. But there was no relevant effect on HERG channel deactivation. The potency for block of HERG channels by droperidol was significantly decreased with mutation of Phe-656 to Thr or mutation of Ser-631 to Ala, respectively. However, mutation of Phe-656 to Met or the double mutation F656M/S631A had no effect on channel sensitivity to block by droperidol. Droperidol potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation. Channel blockade shows greatest affinity for the open and inactivated state. Aromatic residue at position 656 may participate in droperidol binding, and inactivation gating can induce a conformational state that optimizes droperidol binding to the channel.

Hydroxyzine, a First Generation H1-Receptor Antagonist, Inhibits Human Ether-a-go-go–Related Gene (HERG) Current and Causes Syncope in a Patient With the HERG Mutation

QT prolongation, a risk factor for arrhythmias, can result from genetic variants in one (or more) of the genes governing cardiac repolarization as well as intake of drugs known to affect a cardiac K+ channel encoded by human ether-a-go-go–related gene (HERG). In this paper, we will report a case of drug-induced long QT syndrome associated with an H1-receptor antagonist, hydroxyzine, in which a mutation was identified in the HERG gene. After taking 75 mg of hydroxyzine for several days, a 34-year-old female began to experience repetitive syncope. The deleterious effect of hydroxyzine was suspected because QTc interval shortened from 630 to 464 ms after cessation of the drug. Later on, the patient was found to harbor an A614V-HERG mutation. By using the patch-clamp technique in the heterologous expression system, we examined the functional outcome of the A614V mutation and confirmed a dominant-negative effect on HERG expression. Hydroxyzine concentration-dependently inhibited both wild-type (WT) and WT/A614V-HERG K+ currents. Half-maximum block concentrations of WT and WT/A614V-HERG K+ currents were 0.62 and 0.52 μM, respectively. Thus, accidental combination of genetic mutation and intake of hydroxyzine appeared to have led to a severe phenotype, probably, syncope due to torsade de pointes.

Phosphatidylinositol 4,5-bisphosphate interactions with the HERG K+ channel

Regulation of ion channel activity plays a central role in controlling heart rate, rhythm, and contractility responses to cardiovascular demands. Dynamic beat-to-beat regulation of ion channels is precisely adjusted by autonomic stimulation of cardiac G protein-coupled receptors. The rapidly activating delayed rectifier K(+) current (I (Kr)) is produced by the channel that is encoded by human ether-a-gogo-related gene (HERG) and is essential for the proper repolarization of the cardiac myocyte at the end of each action potential. Reduction of I (Kr) via HERG mutations or drug block can lead to lethal cardiac tachyarrhythmias. Autonomic regulation of HERG channels is an area of active investigation with the emerging picture of a complex interplay of signal transduction events, including kinases, second messengers, and protein-protein interactions. A recently described pathway for regulation of HERG is through channel interaction with the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2). Changes in cellular PIP2 concentrations may occur with Gq-coupled receptor activation. Here, we review the evidence for PIP2-HERG interactions, its potential biological significance, and unfilled gaps in our understanding of this regulatory mechanism.

Structural Requirements of Human Ether-a-go-go– related Gene Channels for Block by Bupivacaine

Local anesthetics interact with human ether-a-go-go-related gene (HERG) channels via the aromatic amino acids Y652 and F656 in the S6 region. This study aimed to establish whether the residues T623, S624, and V625 residing deeper within the pore are also involved in HERG channel block by bupivacaine. In addition, the study aimed to further define the role of the aromatic residues Y652 and F656 in bupivacaine inhibition by mutating these residues to threonine. Alanine and threonine mutants were generated by site-directed mutagenesis. Electrophysiologic and pharmacologic properties of wild-type and mutant HERG channels were established using two-electrode voltage-clamp recordings of Xenopus laevis oocytes expressing HERG channels. Tail currents at -120 mV through HERG wild-type channels were inhibited with an IC50 value of 132 +/- 22 microm (n = 33). Bupivacaine (300 microm) inhibited wild-type tail currents by 62 +/- 12% (n = 7). Inhibition of HERG tail currents by bupivacaine (300 microm) was reduced by all mutations (P < 0.001). The effect was largest for F656A (inhibition 5 +/- 2%, n = 6) in the lower S6 region and for T623A (inhibition 13 +/- 4%, n = 9) near the selectivity filter. Introducing threonine at positions 656 and 652 significantly reduced inhibition by bupivacaine compared with HERG wild type (P < 0.001). The authors' results indicate that not only the aromatic residues Y652 and F656 but also residues residing deeper within the pore and close to the selectivity filter of HERG channels are involved in inhibition of HERG channels by the low-affinity blocker bupivacaine.

Inhibition of HERG channels by the local anaesthetic articaine

Articaine is an amide local anaesthetic widely used in dentistry. Human ether-a-go-go-related gene (HERG) potassium channels constitute potential targets involved in cardiotoxic side-effects of various pharmacological agents including amide local anaesthetics. The aim of this study was to determine the sensitivity of HERG channels to the inhibitory action of articaine and to further evaluate the effect of the mutations Y652A and F656A in the putative drug-binding region of HERG on the sensitivity for articaine. We examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by articaine. Whole cell patch-clamp recordings were performed at room temperature. Inhibition of HERG wild-type and HERG Y652A channels by articaine was concentration dependent and reversible. The concentration-response data were described by Hill functions (wild type: IC50 = 224 +/- 6 micromol L-1, Hill coefficient h = 1.17 +/- 0.03, n = 23; Y652A: IC50 = 360 +/- 48 micromol L-1, h = 0.93 +/- 0.08, n = 26). The mutation Y5652A decreased the sensitivity by factor 1.6. The mutation F656A decreased inhibition of inward tail currents by 300 micromol L-1 articaine in 100 mmol extracellular K+ 3-fold. Our results indicate that the local anaesthetic articaine does not inhibit HERG channels at clinically relevant concentrations. Articaine may therefore constitute a safer alternative for local and regional anaesthesia. The aromatic amino acid F656 rather than Y652 in the S6 region might play a role in interaction of the drug with the channel.

Utility of Hair Clips for Scalp Surgeries

Background and objective: Articaine is an amide local anaesthetic widely used in dentistry. Human ether-a-go-go-related gene (HERG) potassium channels constitute potential targets involved in cardiotoxic side-effects of various pharmacological agents including amide local anaesthetics. The aim of this study was to determine the sensitivity of HERG channels to the inhibitory action of articaine and to further evaluate the effect of the mutations Y652A and F656A in the putative drug-binding region of HERG on the sensitivity for articaine. Methods: We examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by articaine. Whole cell patch-clamp recordings were performed at room temperature. Results: Inhibition of HERG wild-type and HERG Y652A channels by articaine was concentration dependent and reversible. The concentration-response data were described by Hill functions (wild type: IC50 = 224 ± 6 μmol L−1, Hill coefficient h = 1.17 ± 0.03, n = 23; Y652A: IC50 = 360 ± 48 μmol L−1, h = 0.93 ± 0.08, n = 26). The mutation Y5652A decreased the sensitivity by factor 1.6. The mutation F656A decreased inhibition of inward tail currents by 300 μmol L−1 articaine in 100 mmol extracellular K+ 3-fold. Conclusions: Our results indicate that the local anaesthetic articaine does not inhibit HERG channels at clinically relevant concentrations. Articaine may therefore constitute a safer alternative for local and regional anaesthesia. The aromatic amino acid F656 rather than Y652 in the S6 region might play a role in interaction of the drug with the channel.

A novel splice mutation of HERG in a Chinese family with long QT syndrome

Congenital long QT syndrome (LQTS) is a genetically heterogeneous disease in which six ion-channel genes have been identified. The phenotype-genotype relationships of the HERG (human ether-a-go-go-related gene) mutations are not fully understood. The objective of this study is to identify the underlying genetic basis of a Chinese family with LQTS and to characterize the clinical manifestations properties of the mutation. Single strand conformation polymorphism (SSCP) analyses were conducted on DNA fragments amplified by polymerase chain reaction from five LQT-related genes. Aberrant conformers were analyzed by DNA sequencing. A novel splice mutation in C-terminus of HERG was identified in this Chinese LQTS family, leading to the deletion of 11-bp at the acceptor splice site of Exon9 [Exon9 IVS del (-12-->-2)]. The mutation might affect, through deficient splicing, the putative cyclic nucleotide binding domain (CNBD) of the HERG K(+) channel. This mutation resulted in a mildly affected phenotype. Only the proband had a history of syncopes, while the other three individuals with long QT interval had no symptoms. Two other mutation carriers displayed normal phenotype. No sudden death occurred in the family. The 4 affected individuals and the two silent mutation carriers were all heterozygous for the mutation. It is the first splice mutation of HERG reported in Chinese LQTS families. Clinical data suggest that the CNBD mutation may be less malignant than mutations occurring in the pore region and be partially dominant over wild-type function.

Local Anesthetic Interaction with Human Ether-a-go-go –related Gene (HERG) Channels

Human ether-a-go-go-related gene (HERG) potassium channels constitute a potential target involved in cardiotoxic side effects of amino-amide local anesthetics. The molecular interaction site of these low-affinity blockers with HERG channels is currently unknown. The aim of this study was to determine the effect of the mutations Y652A and F656A in the putative drug binding region of HERG on the inhibition by bupivacaine, ropivacaine, and mepivacaine. The authors examined the inhibition of wild-type and mutant HERG channels, transiently expressed in Chinese hamster ovary cells by bupivacaine, ropivacaine, and mepivacaine. Whole cell patch clamp recordings were performed at room temperature. Inhibition of HERG wild-type and mutant channels by the different local anesthetics was concentration dependent, stereoselective, and reversible. The sensitivity decreased in the order bupivacaine > ropivacaine > mepivacaine for wild-type and mutant channels. The mutant channels were approximately 4-30 times less sensitive to the inhibitory action of the different local anesthetics than the wild-type channel. The concentration-response data were described by Hill functions (bupivacaine: wild-type IC50 = 22 +/- 2 microm, n = 38; Y652A IC50 = 95 +/- 5 microm, n = 31). The mutations resulted in a change of the stereoselectivity of HERG channel block by ropivacaine. The potency of the local anesthetics to inhibit wild-type and mutant channels correlated with the lipophilicity of the drug (r > 0.9). These results indicate that local anesthetics specifically but not exclusively interact with the aromatic residues Y652 and F656 in S6 of HERG channels.

HERG Channel (Dys)function Revealed by Dynamic Action Potential Clamp Technique

The human ether-a-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier potassium current ( I Kr). Per-Arnt-Sim domain mutations of the HERG channel are linked to type 2 long-QT syndrome. We studied wild-type and/or type 2 long-QT syndrome-associated mutant (R56Q) HERG current ( I HERG) in HEK-293 cells, at both 23 and 36°C. Conventional voltage-clamp analysis revealed mutation-induced changes in channel kinetics. To assess functional implication(s) of the mutation, we introduce the dynamic action potential clamp technique. In this study, we effectively replace the native I Kr of a ventricular cell (either a human model cell or an isolated rabbit myocyte) with I HERG generated in a HEK-293 cell that is voltage-clamped by the free-running action potential of the ventricular cell. Action potential characteristics of the ventricular cells were effectively reproduced with wild-type I HERG, whereas the R56Q mutation caused a frequency-dependent increase of the action potential duration in accordance with the clinical phenotype. The dynamic action potential clamp approach also revealed a frequency-dependent transient wild-type I HERG component, which is absent with R56Q channels. This novel electrophysiological technique allows rapid and unambiguous determination of the effects of an ion channel mutation on the ventricular action potential and can serve as a new tool for investigating cardiac channelopathies.

Novel C-terminus frameshift mutation, 1122fs/147, of HERG in LQT2: additional amino acids generated by frameshift cause accelerated inactivation

Objective. – The function of the C-terminus region of the human ether-a-go-go related gene (HERG) has not been well characterized except for its involvement in trafficking. To understand further the role of C-terminus region, we performed a functional analysis of a novel frameshift mutation (1122fs/147) identified in a Japanese long QT syndrome 2 (LQT2) patient who had recurrent episodes of syncope. Methods. – Wild type (WT) and mutant HERG plasmids were transfected into human embryonic kidney (HEK-293) cells, and whole-cell current was recorded by the patch-clamp technique. Confocal microscopy was performed to examine the membrane distribution of channel protein using a green fluorescent protein tagged to the N-terminus of HERG. Results. – The mutant 1122fs/147 alone could express current, but reduced density by 74% of control. No dominant negative effect was noted with co-expression of WT and 1122fs/147. Activation and deactivation time constants were not changed, while inactivation was accelerated in 1122fs/147 compared to WT, and V 1/2 of steady-state inactivation curve shifted by 11 mV in the negative direction. Current density of 1123stop mutant revealed 49% reduction compared to WT and showed no shift in steady-state inactivation. Confocal microscopy revealed reduced protein expression on the cell surface both in 1122fs/147 and 1123stop mutants compared to WT. Conclusion. – Frameshift mutation at the C-terminus region with additional 147 amino acids evoked a loss of function of the HERG channel. A negative shift in steady-state inactivation induced by the additional 147 amino acids and trafficking defect contribute to a reduced current amplitude of 1122fs/147.

High affinity HERG K + channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K + channels by structurally and therapeutically diverse drugs is associated with the ‘acquired’ form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current ( I HERG) blockade at 37 ± 1 °C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I HERG tails, measured at −40 mV following activating pulses to +30 mV, were blocked with IC 50 values of ∼59 and 70 nM for DRONED and AMIOD, respectively. I HERG inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K +] (94 mM) reduced the potency of I HERG inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I HERG can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.

Defective assembly and trafficking of mutant HERG channels with C-terminal truncations in long QT syndrome

Mutations in the human ether-a-go-go-related gene ( HERG) cause long QT syndrome type 2 (LQT2). HERG encodes a voltage-gated potassium channel consisting of four subunits. Tetrameric assembly is required for the formation of functional HERG channels. In the present work, we studied the role of assembly in HERG channel dysfunction of LQT2 mutations Q725X and R1014X, both of which cause truncations of the C-terminus of HERG channels. When expressed in HEK293 cells, Q725X did not generate HERG current, while R1014X generated HERG current with markedly reduced amplitude. Western blot analysis showed that both mutations caused defective trafficking of HERG channel proteins. Using sucrose gradient centrifugation we showed that wild type HERG and R1014X formed a tetrameric structure, whereas Q725X was expressed as a monomer. When coexpressed with wild type HERG, R1014X, but not Q725X, caused dominant negative suppression of wild type HERG current. Coimmunoprecipitation experiments showed that the lack of dominant negative effect by Q725X was due to failure of mutant subunits to coassemble with wild type subunits. These results suggest that the Q725X mutation causes HERG channel dysfunction by disruption of tetrameric assembly of HERG channels. In contrast, the R1014X mutation is capable of forming tetrameric structure, and it causes HERG channel dysfunction by defective trafficking of the mutant protein.

In vivo identification of genes that modify ether-a-go-go-related gene activity in Caenorhabditis elegans may also affect human cardiac arrhythmia.

Human ether-a-go-go-related gene (HERG) encodes the pore-forming subunit of I(Kr), a cardiac K(+) channel. Although many commonly used drugs block I(Kr), in certain individuals, this action evokes a paradoxical life-threatening cardiac rhythm disturbance, known as the acquired long QT syndrome (aLQTS). Although aLQTS has become the leading cause of drug withdrawal by the U.S. Food and Drug Administration, DNA sequencing in aLQTS patients has revealed HERG mutations only in rare cases, suggesting that unknown HERG modulators are often responsible. By using the worm Caenorhabditis elegans, we have developed in vivo behavioral assays that identify candidate modulators of unc-103, the worm HERG orthologue. By using RNA-interference methods, we have shown that worm homologues of two HERG-interacting proteins, Hyperkinetic and K channel regulator 1 (KCR1), modify unc-103 function. Examination of the human KCR1 sequence in patients with drug-induced cardiac repolarization defects revealed a sequence variation (the substitution of isoleucine 447 by valine, I447V) that occurs at a reduced frequency (1.1%) relative to a matched control population (7.0%), suggesting that I447V may be an allele for reduced aLQTS susceptibility. This clinical result is supported by in vitro studies of HERG dofetilide sensitivity by using coexpression of HERG with wild-type and I447V KCR1 cDNAs. Our studies demonstrate the feasibility of using C. elegans to assay and potentially identify aLQTS candidate genes.

Structural determinants of HERG channel block by clofilium and ibutilide.

Block of human ether-a-go-go related gene (HERG) K(+) channels by a variety of medications has been linked to acquired long QT syndrome, a disorder of cardiac repolarization that predisposes to lethal arrhythmias. The drug-binding site is composed of residues that face into the central cavity of the channel. Two aromatic residues located on the S6 domain (Tyr652 and Phe656) are particularly important structural determinants of drug block. The role of pore helix residues (Thr623, Ser624, Val625) is less clear. In this study, we compared the pharmacological properties of two structurally related compounds, ibutilide and clofilium. Both compounds are charged amines with a single phenyl ring. Clofilium, a chlorobenzene derivative, is a potent blocker of HERG channels, but has a remarkably slower time course for recovery from block than ibutilide, a methanesulfonanilide. The difference in the rate of recovery from block can be explained simply by variation in drug trapping. There is little recovery from clofilium block with D540K HERG channels that permit untrapping at hyperpolarized potentials. Alanine-scanning mutagenesis of the S6 domain and a portion of the pore helix revealed that the binding site residues were the same for both compounds. However, S624A, located at the base of the pore helix, was the only HERG mutation that enabled rapid recovery from clofilium block. In summary, the pore helix residues are important components of the HERG drug binding site, and may be particularly important for drugs with polar substituents, such as a halogen (e.g., clofilium) or a methanesulfonamide (e.g., ibutilide).

Pharmacological rescue of trafficking defective HERG channels formed by coassembly of wild-type and long QT mutant N470D subunits

Mutations in the human ether-a-go-go-related gene (HERG) cause long QT syndrome. We previously showed that the HERG N470D mutation expressed as homotetrameric channels causes a protein trafficking defect, and this can be corrected by the HERG channel blocking drug E-4031. The N470D mutant also has been reported to cause dominant negative suppression of HERG current when coexpressed with wild-type channel subunits. The aims of this study were 1). to investigate the molecular mechanism responsible for the dominant negative effect of the N470D mutant coexpressed with wild-type subunits and 2). to test whether the trafficking defective heteromeric channels could be pharmacologically rescued by E-4031. Using a combination of immunoprecipitation and Western blot methods, we showed that N470D mutant and wild-type HERG subunits were physically associated in the endoplasmic reticulum as heteromeric channels. The coassembly resulted in the retention of both wild-type and N470D subunits in the endoplasmic reticulum. Culturing cells in E-4031 increased the cell surface expression of these channels, although with an altered electrophysiological phenotype. These results suggest that the dominant negative effect of the N470D wild-type coassembled channels is caused by retention of heteromeric channels in the endoplasmic reticulum and that the trafficking defect of these channels can be corrected by specific pharmacological strategies.