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

Extracellular K+ concentration controls cell surface density of IKr in rabbit hearts and of the HERG channel in human cell lines

Although the modulation of ion channel gating by hormones and drugs has been extensively studied, much less is known about how cell surface ion channel expression levels are regulated. Here, we demonstrate that the cell surface density of both the heterologously expressed K+ channel encoded by the human ether-a-go-go-related gene (HERG) and its native counterpart, the rapidly activating delayed rectifier K+ channel (IKr), in rabbit hearts in vivo is precisely controlled by extracellular K+ concentration ([K+]o) within a physiologically relevant range. Reduction of [K+]o led to accelerated internalization and degradation of HERG channels within hours. Confocal analysis revealed colocalization between HERG and ubiquitin during the process of HERG internalization, and overexpression of ubiquitin facilitated HERG degradation under low [K+]o. The HERG channels colocalized with a marker of multivesicular bodies during internalization, and the internalized HERG channels were targeted to lysosomes. Our results provide the first evidence to our knowledge that the cell surface density of a voltage-gated K+ channel, HERG, is regulated by a biological factor, extracellular K+. Because hypokalemia is known to exacerbate long QT syndrome (LQTS) and Torsades de pointes tachyarrhythmias, our findings provide a potential mechanistic link between hypokalemia and LQTS.

Endocytic control of ion channel density as a target for cardiovascular disease

Ion channels encoded by the human ether-a-go-go-related gene (HERG) give rise to the rapidly activating delayed rectifier K+ current (IKr), the perturbation of which causes ventricular arrhythmias associated with inherited and acquired long QT syndrome. Electrolyte imbalances, such as reduced serum K+ levels (hypokalemia), also trigger these potentially fatal arrhythmias. In this issue of the JCI, Guo et al. report that physiological levels of serum K+ are required to maintain normal HERG surface density in HEK 293 cells and IKr in rabbit cardiomyocytes. They found that hypokalemia evoked HERG channel ubiquitination, enhanced internalization via endocytosis, and ultimately degradation at the lysosome, thus identifying unbridled turnover as a mechanism of hypokalemia-induced arrhythmia. But too little channel turnover can also cause disease, as suggested by Kruse et al. in a study also in this issue. The authors identified mutations in TRPM4--a nonselective cation channel--in a large family with progressive familial heart block type I and showed that these mutations prevented channel internalization (see the related articles beginning on pages 2745 and 2737, respectively).

Extracellular potassium dependency of block of HERG by quinidine and cisapride is primarily determined by the permeant ion and not by inactivation.

Drug induced Long QT syndrome results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases prolongation of the QT interval can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with abnormal serum electrolyte levels due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, alcoholism, and aging. Extracellular cations have significant influence on HERG channel gating and in some instances they have been shown to alter drug block of HERG. However, the mechanisms by which drug block is altered in different extracellular cation solutions are not well understood. In this study, HERG block by quinidine and cisapride was assessed in extracellular solutions of calcium, potassium, rubidium, cesium, and tetraethylammonium (TEA) using two-electrode voltage clamping of Xenopus oocytes. Consistent with previous reports we show that increases in extracellular potassium reduce HERG block by quinidine and cisapride. We also show that increasing extracellular rubidium and cesium reduced HERG block by quinidine and cisapride whereas increasing extracellular calcium and extracellular TEA did not alter HERG block by quinidine and cisapride. These results demonstrate that at lower extracellular potassium concentrations, the permeant ion is almost exclusively responsible for the reduction in quinidine and cisapride block of HERG due to increases in extracellular potassium.

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.

Blockade of HERG K+ channel by isoquinoline alkaloid neferine in the stable transfected HEK293 cells

We studied the effects of isoquinoline alkaloid neferine (Nef) extracted from the seed embryo of Nelumbo nucifera Gaertn on Human ether-à-go-go-related gene (HERG) channels stably expressed in human embryonic kidney (HEK293) cells using whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. Nef induced a concentration-dependent decrease in current amplitude according to the voltage steps and tail currents of HERG with an IC(50) of 7.419 microM (n(H) -0.5563). Nef shifted the activation curve in a significantly negative direction and accelerated recovery from inactivation and onset of inactivation, however, slowed deactivation. In addition, it had no significant influence on steady-state inactivation curve. Western blot and immunofluorescence results suggested Nef had no significant effect on the expression of HERG protein. In summary, Nef can block HERG K(+) channels that functions by changing the channel activation and inactivation kinetics. Nef has no effect on the generation and trafficking of HERG protein. A blocked-off HERG channel was one mechanism of the anti-arrhythmic effects by Nef.

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.

Androgens Stabilize HERG Potassium Channel Protein Via Stimulation Of Androgen Receptor Variant Ar45

Proarrhythmic drugs induce long QT syndrome more frequently in women than men. The present study was designed to determine whether androgens regulate the function and expression of the human ether-a-go-go-related gene (HERG) encoded K+ channel, which is largely responsible for determining the QT interval. In a concentration-dependent manner (10−9 to 10−6 M for 24 h), 5α-dihydrotestosterone (5α-DHT) increased HERG protein abundance in HEK293 cells stably expressing HERG in the presence of co-expressed cardiac androgen receptor variant (AR45). The elevation of HERG protein was seen in ER, Golgi and plasma membrane without clear preferential colocalization. Co-expression of the more common form of the androgen receptor did not confer 5α-DHT augmentation of HERG protein. Proteasome inhibitors, N-acetyl-L-leucyl-L-leucyl-L-norleucinal and MG132 prevented the 5α-DHT-dependent enhancement of HERG as did the lysosome inhibitor, bafilomycin A1. Consistently, the cycloheximide-based protein chase study showed that 5α-DHT prolonged HERG protein half-life. 5α-DHT/AR45 signaling induced phosphorylation of extracellular signaling regulated kinase (ERK1/2). Blockade of ERK1/2 with PD98059 and U0126 prevented the effect of androgen on HERG protein abundance. Functional studies showed that 5-DHT treatment for 24 h increased HERG K+ current density in CHO cells co-transfected with cDNAs of AR45 and HERG channels. Moreover, 5α-DHT also increased ERG protein abundance in isolated rabbit cardiac myocytes. In conclusion, these data provide evidence that stimulation of AR45 receptors by androgens upregulates HERG K+ channel abundance and activity mainly through stabilizing HERG protein in an ERK1/2 dependent mechanism and suggest a mechanism to explain the sex difference in the long QT syndrome.

The Effects of a Novel Anti‐arrhythmic Drug, Acehytisine Hydrochloride, on the Human Ether‐a‐go‐go Related Gene K+ Channel and Its Trafficking

Many drugs cause severe side-effects such as long QT syndromes by blocking the human ether-a-go-go related gene (HERG) K(+) channels and/or disrupting HERG protein trafficking. Acehytisine hydrochloride is an anti-arrhythmic drug in phase IV clinical trial. To study whether acehytisine hydrochloride affects HERG channel activity and protein trafficking, we expressed HERG in human embryonic kidney 293 (HEK293) cells and recorded HERG channel currents with a whole-cell patch clamp technique. We also measured the protein levels by Western blot analysis. We found that acehytisine hydrochloride inhibited HERG step current (I(HERG)) in a concentration-dependent manner. However, it had little effect on the tail current (I(tail)). In addition, acehytisine hydrochloride accelerated channel inactivation and slowed recovery from inactivation. In contrast, it did not inhibit HERG protein (135 and 155 kD) trafficking, although it reduced the 155 kD band density at 2500 microM. Moreover, the F656C mutation in the S6 domain abolished acehytisine hydrochloride inhibition on the I(HERG) and enhanced the inhibitive effects on the trafficking of the 155 kD band. Acehytisine hydrochloride interrupted HERG protein trafficking at 1000 and 2500 microM. Our data showed that acehytisine hydrochloride could inhibit I(HERG), but had no effects on I(tail) until the concentration was above 1000 microM. Therefore, acehytisine hydrochloride may not induce QT interval prolongation and could be a promising anti-arrhythmic drug without severe side-effects.

Inhibition of the Human Ether-a-go-go-related Gene (HERG) K+ Channels by Lindera erythrocarpa

Lindera erythrocarpa Makino (Lauraceae) is used as a traditional medicine for analgesic, antidote, and antibacterial purposes and shows anti-tumor activity. We studied the effects of Lindera erythrocarpa on the human ether-a-go-go-related gene (HERG) channel, which appears of importance in favoring cancer progression in vivo and determining cardiac action potential duration. Application of MeOH extract of Lindera erythrocarpa showed a dose-dependent decrease in the amplitudes of the outward currents measured at the end of the pulse (IHERG) and the tail currents of HERG (Itail). When the BuOH fraction and H2O fraction of Lindera erythrocarpa were added to the perfusate, both IHERG and Itail were suppressed, while the hexane fraction, CHCl3 fraction, and EtOAc fraction did not inhibit either IHERG or Itail. The potential required for half-maximal activation caused by EtOAc fraction, BuOH fraction, and H2O fraction shifted significantly. The BuOH fraction and H2O fraction (100 µg/mL) decreased gmax by 59.6% and 52.9%, respectively. The H2O fraction- and BuOH fraction-induced blockades of Itail progressively decreased with increasing depolarization, showing the voltage-dependent block. Our findings suggest that Lindera erythrocarpa, a traditional medicine, blocks HERG channel, which could contribute to its anticancer and cardiac arrhythmogenic effect.

Participation of HERG channel cytoplasmic structures on regulation by the G protein-coupled TRH receptor

Human ether-a-go-go-related gene (HERG) channels heterologously expressed in Xenopus oocytes are regulated by the activation of G protein-coupled hormone receptors that, like the thyrotropin-releasing hormone (TRH) receptor, activate phospholipase C. Previous work with serially deleted HERG mutants suggested that residues 326-345 located in the proximal domain of the channels amino terminus might be required for the hormonal modulation of HERG activation. Generation of new channel mutants deleted in this region further point to the amino acid sequence between residues 326 and 332 as a possible determinant of the TRH effects, but individual or combined single-point mutations in this sequence demonstrate that maintenance of its consensus sites for phosphorylation and/or interaction with regulatory components is not important for the modulatory response(s). The TRH-induced effects also remained unaltered when a basic amino acid cluster located between residues 362 and 366 is eliminated. Additionally, no effect of TRH was observed in channels carrying single-point mutations at the beginning of the intracellular loop linking transmembrane domains S4 and S5. Our results indicate that a correct structural arrangement of the amino terminal domains is essential for the hormone-induced modifications of HERG activation. They also suggest that the hormonal regulatory action is transmitted to the transmembrane channel core through interactions between the cytoplasmic domains and the initial portion of the S4-S5 linker.

Emerging roles for ether‐á‐go‐go‐related gene potassium channels in the brain

Emerging roles for ether‐á‐go‐go‐related gene potassium channels in the brain

Therapeutic Strategies for Long-QT Syndrome

The discovery of genetic defects underlying long-QT syndrome (LQTS) has allowed to identify important genotype-phenotype correlations that are now being used for risk stratification. The next challenge is to exploit the new information on the pathophysiology of the disease derived from molecular genetics to devise more effective therapies. The successful response of LQT1 patients to β-blockers, the QT-shortening action of sodium channel blockers in at least some LQT3 patients, and the importance of maintaining adequate plasma potassium levels in LQT2 patients clearly demonstrate the importance of selecting therapy in the context of the molecular substrate. The hurdles on the road toward the development of novel therapeutic strategies are represented by the variable expressivity of the disease, the high prevalence of “private” mutations, and the role of genetic and nongenetic factors that act as modifiers of the phenotype. Given the large number of mutations identified and their phenotypic complexity, it is clearly impossible to anticipate a scenario in which each mutation will be managed through a specific therapy. However, the evidence that mutations in the LQTS gene may be clustered based on their electrophysiological profile and on their response to specific drugs may provide the rationale for the development of mutation-specific therapies. In this article, we will review the most relevant genotype-phenotype features of LQTS and the strategies explored to develop novel therapeutic approaches. ### Definition and Prevalence The congenital LQTS is an inherited arrhythmogenic disease characterized by abnormally prolonged QT interval leading to life-threatening arrhythmias in the presence of a structurally normal heart.1 Different clinical variants of LQTS have been identified. Romano Ward syndrome, transmitted as an autosomal dominant trait, is the most common form of LQTS and presents only a cardiac phenotype. Less prevalent variants of LQTS, such as Andersen and Timothy syndromes or the recessive Jervell and Lange-Nielsen syndromes, also present …

Electropharmacological properties of telmisartan in blocking hKv1.5 and HERG potassium channels expressed onXenopus laevisoocytes1

The objectives of this study were to investigate the inhibitory action of telmisartan, a selective angiotensin II type 1 receptor antagonist, on hKv1.5 and human ether-a-go-go-related gene (HERG) channels expressed on Xenopus laevis oocytes. hKv1.5 and HERG channels were expressed on Xenopus laevis oocytes and studied using the 2-microelectrode voltage clamp technique. In hKv1.5 channels, telmisartan produced a voltage- and concentration-dependent inhibition; the efficacies of blockade were different at peak and 1.5 s end-pulse currents, which were 7.75%+/-2.39% (half-maximal inhibition concentration [IC50]=2.25+/-0.97 micromol/L) and 52.64%+/-3.77% (IC50=0.82+/-0.39 micromol/L) at 1 micromol/L telmisartan, respectively. Meanwhile, telmisartan accelerated the inactivation of the channels. However, telmisartan exhibited a low affinity for HERG channels (IC50=24.35+/-5.06 micromol/L); the blockade was voltage- and concentration-dependent. Telmisartan preferentially blocked open-state HERG channels. The slow time constants of deactivation were accelerated (n=6, P<0.05), which was inconsistent with the "foot-in-the-door"effect. Telmisartan blocks hKv1.5 potassium channels involving open and inactivated states at plasma concentration levels of therapeutic doses; whereas the blockade of HERG channels occurs only at supra plasma concentration levels of therapeutic doses and preferentially in open and closed-state channels.

FRET with multiply labeled HERG K+ channels as a reporter of the in vivo coarse architecture of the cytoplasmic domains

The intracellular N-terminus of human ether-a-go-go-related gene (HERG) potassium channels constitutes a key determinant of activation and deactivation characteristics and is necessary for hormone-induced modifications of gating properties. However, the general organization of the long amino and carboxy HERG terminals remains unknown. In this study we performed fluorescence resonance energy transfer (FRET) microscopy with a library of fluorescent HERG fusion proteins obtained combining site-directed and transposon-based random insertion of GFP variants into multiple sites of HERG. Determinations of FRET efficiencies with functional HERG channels labeled in different combinations localize the fluorophores, introduced in the amino and carboxy ends, in two quadratic planes of 7.8 and 8.6 nm lateral size, showing a vertical separation of nearly 8 nm without major angular torsion between the planes. Similar analysis using labels at positions 345 and 905 of the amino and carboxy terminals, located them slightly above the planes delimited by the amino and carboxy end labels, respectively. Our data also indicate an almost vertical arrangement of the fluorophores introduced in the NH2 and COOH ends and at position 905, but a near 45° angular rotation between the planes delimited by these labels and the 345-located fluorophores. Systematic triangulation using interfluorophore distances coming from multiply labeled channels provides an initial constraint on the overall in vivo arrangement of the HERG cytoplasmic domains, suggesting that the C-linker/CNBD region of HERG hangs centrally below the transmembrane core, with the initial portion of the amino terminus around its top and side surfaces directed towards the gating machinery.

Rapid Kinetic Interactions of Ranolazine With HERG K+ Current

Ranolazine, an anti-ischemic agent, inhibits I Kr [encoded by the human ether-a-go-go-related gene (HERG)] and causes a small QT interval prolongation without any proarrhythmic events. The objective of this study was to elucidate the biophysical characteristics of inhibition of HERG K+ current (IHERG) by ranolazine. We investigated the effects of ranolazine using voltage-clamp and Western blot analyses of HERG channels stably expressed in HEK293 cells. Ranolazine reduced IHERG with the half-maximal inhibitory concentration of 12.0 microM. Block of IHERG by ranolazine was reversible and voltage-dependent but frequency-independent. At 0 mV, the time constants for development of block were 76.6 +/- 1.6, 35.8 +/- 2.4, and 19.4 +/- 1.7 msec with 10, 30, and 100 microM ranolazine (n = 4), respectively. The apparent dissociation constant estimated from the time course of ranolazine-induced IHERG decay was 22.5 microM. After repolarization at -80 and -100 mV, IHERG recovery from ranolazine block followed a monophasic time course with tau values of 204.3 +/- 51.5 and 155.0 +/- 31.9 msec (n = 5), respectively. Intracellular but not extracellular application of a membrane-impermeable (permanently charged) ranolazine analogue caused rapid block of IHERG. Ranolazine did not alter HERG protein trafficking to the plasma membrane. In conclusion, ranolazine caused a time- and voltage-dependent, but frequency-independent, block of IHERG. The kinetics of IHERG inhibition (at positive potentials) and unblock (upon hyperpolarization) by ranolazine were rapid. These distinct and rapid kinetic interactions of ranolazine with IHERG may partially contribute to the observations that the drug is not proarrhythmic despite causing a small prolongation of action potentials and QT intervals.

Thermodynamic and Kinetic Properties of Amino-Terminal and S4-S5 Loop HERG Channel Mutants under Steady-State Conditions

Gating kinetics and underlying thermodynamic properties of human ether-a-go-go-related gene (HERG) K+ channels expressed in Xenopus oocytes were studied using protocols able to yield true steady-state kinetic parameters. Channel mutants lacking the initial 16 residues of the amino terminus before the conserved eag/PAS region showed significant positive shifts in activation voltage dependence associated with a reduction of zg values and a less negative ΔGo, indicating a deletion-induced displacement of the equilibrium toward the closed state. Conversely, a negative shift and an increased ΔGo, indicative of closed-state destabilization, were observed in channels lacking the amino-terminal proximal domain. Furthermore, accelerated activation and deactivation kinetics were observed in these constructs when differences in driving force were considered, suggesting that the presence of distal and proximal amino-terminal segments contributes in wild-type channels to specific chemical interactions that raise the energy barrier for activation. Steady-state characteristics of some single point mutants in the intracellular loop linking S4 and S5 helices revealed a striking parallelism between the effects of these mutations and those of the amino-terminal modifications. Our data indicate that in addition to the recognized influence of the initial amino-terminus region on HERG deactivation, this cytoplasmic region also affects activation behavior. The data also suggest that not only a slow movement of the voltage sensor itself but also delaying its functional coupling to the activation gate by some cytoplasmic structures possibly acting on the S4-S5 loop may contribute to the atypically slow gating of HERG.

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.

Electrophysiological and Fluorescence Microscopy Studies with HERG Channel/EGFP Fusion Proteins

HERG (human ether-a-go-go-related gene) encodes the Kv11.1 protein alpha-subunit that underlies the rapidly activating delayed rectifier K+ current (IKr) in the heart. Alterations in the functional properties or membrane incorporation of HERG channels, either by genetic mutations or by administration of drugs, play major roles in the development of life-threatening torsades de pointes cardiac arrhythmias. Visualization of ion channel localization is facilitated by enhanced green fluorescent protein (EGFP) tagging, but this process can alter their properties. The aim of the present study was to characterize the electrophysiological properties and the cellular localization of HERG channels in which EGFP was tagged either to the C terminus (HERG/EGFP) or to the N terminus (EGFP/HERG). These fusion constructs were transiently expressed in human embryonic kidney (HEK) 293 cells, and the whole-cell patch-clamp configuration and a confocal laser scanning microscope with primary anti-HERG antibodies and fluorescently labeled secondary antibodies were used. For EGFP/HERG channels the deactivation kinetics were faster and the peak tail current density was reduced when compared to both wild-type HERG channels and HERG/EGFP channels. Laser scanning microscopic studies showed that both fusion proteins were localized in the cytoplasm and on discrete microdomains in the plasma membrane. The extent of labeling with anti-HERG antibodies of HEK 293 cells expressing EGFP/HERG channels was less when compared to HERG/EGFP channels. In conclusion, both electrophysiological and immunocytochemical studies showed that EGFP/HERG channels themselves have a protein trafficking defect. HERG/EGFP channels have similar properties as untagged HERG channels and, thus, might be especially useful for fluorescence microscopy studies.

HERG K+ Channel Blockade by the Novel Antiviral Drug Sophocarpine

Human ether-à-go-go-related gene (HERG) encodes the rapid component of the cardiac delayed rectifier K+ current, which has an important role in the repolarization of the cardiac action potential. QT interval prolongation through HERG channel inhibition is associated with a risk of torsade de pointes arrhythmias and is a major challenge for drug development. The effects of the novel antiviral drug sophocarpine (SC) were examined on stably expressed HERG channels in human embryonic kidney (HEK293) cells using a whole-cell patch clamp technique, Western blot analysis and immunofluorescence experiments. SC inhibited HERG channels in a concentration-dependent manner, with an IC50 of 100-300 microM. SC significantly accelerated channel inactivation, recovery from inactivation and onset of inactivation. In addition, it had no effect on channel activation and deactivation. Based on Western blot and immunofluorescence results, SC had no significant effect on the expression of HERG protein. In summary, SC is a potent blocker of HERG K+ channels that functions by changing the channel inactivation kinetics. In addition, SC has no effect on the generation and trafficking of HERG protein.