hERG1a N-terminal eag domain-containing polypeptides regulate homomeric hERG1b and heteromeric hERG1a/hERG1b channels: a possible mechanism for long QT syndrome

Human ether-á-go-go-related gene (hERG) potassium channels are critical for cardiac action potential repolarization. Cardiac hERG channels comprise two primary isoforms: hERG1a, which has a regulatory N-terminal Per-Arnt-Sim (PAS) domain, and hERG1b, which does not. Isolated, PAS-containing hERG1a N-terminal regions (NTRs) directly regulate NTR-deleted hERG1a channels; however, it is unclear whether hERG1b isoforms contain sufficient machinery to support regulation by hERG1a NTRs. To test this, we constructed a series of PAS domain-containing hERG1a NTRs (encoding amino acids 1-181, 1-228, 1-319, and 1-365). The NTRs were also predicted to form from truncation mutations that were linked to type 2 long QT syndrome (LQTS), a cardiac arrhythmia disorder associated with mutations in the hERG gene. All of the hERG1a NTRs markedly regulated heteromeric hERG1a/hERG1b channels and homomeric hERG1b channels by decreasing the magnitude of the current-voltage relationship and slowing the kinetics of channel closing (deactivation). In contrast, NTRs did not measurably regulate hERG1a channels. A short NTR (encoding amino acids 1-135) composed primarily of the PAS domain was sufficient to regulate hERG1b. These results suggest that isolated hERG1a NTRs directly interact with hERG1b subunits. Our results demonstrate that deactivation is faster in hERG1a/hERG1b channels compared to hERG1a channels because of fewer PAS domains, not because of an inhibitory effect of the unique hERG1b NTR. A decrease in outward current density of hERG1a/hERG1b channels by hERG1a NTRs may be a mechanism for LQTS.     

The N-linker region of hERG1a upregulates hERG1b potassium channels

A major physiological role of hERG1 (human Ether-á-go-go-Related Gene 1) potassium channels is to repolarize cardiac action potentials. Two isoforms, hERG1a and hERG1b, associate to form the potassium current I_Kr in cardiomyocytes. Inherited mutations in hERG1a or hERG1b cause prolonged cardiac repolarization, long QT syndrome, and sudden death arrhythmia. hERG1a subunits assemble with and enhance the number of hERG1b subunits at the plasma membrane, but the mechanism for the increase in hERG1b by hERG1a is not well understood. Here, we report that the hERG1a N-terminal region expressed in trans with hERG1b markedly increased hERG1b currents and increased biotin-labeled hERG1b protein at the membrane surface. hERG1b channels with a deletion of the N-terminal 1b domain did not have a measurable increase in current or biotinylated protein when coexpressed with hERG1a N-terminal regions, indicating that the 1b domain was required for the increase in hERG1b. Using a biochemical pull-down interaction assay and a FRET hybridization experiment, we detected a direct interaction between the hERG1a N-terminal region and the hERG1b N-terminal region. Using engineered deletions and alanine mutagenesis, we identified a short span of amino acids at positions 216 to 220 within the hERG1a “N-linker” region that were necessary for the upregulation of hERG1b. We propose that direct structural interactions between the hERG1a N-linker region and the hERG1b 1b domain increase hERG1b at the plasma membrane. Mechanisms regulating hERG1a and hERG1b are likely critical for cardiac function, may be disrupted by long QT syndrome mutants, and serve as potential targets for therapeutics.     

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

The hERG (human ether-a-go-go-related gene) encodes the α subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). Dysfunction of hERG channels due to mutations or certain medications causes long QT syndrome, which can lead to fatal ventricular arrhythmias or sudden death. Although the abundance of hERG in the plasma membrane is a key determinant of hERG functionality, the mechanisms underlying its regulation are not well understood. In the present study, we demonstrated that overexpression of the stress-responsive serum- and glucocorticoid-inducible kinase (SGK) isoforms SGK1 and SGK3 increased the current and expression level of the membrane-localized mature proteins of hERG channels stably expressed in HEK 293 (hERG-HEK) cells. Furthermore, the synthetic glucocorticoid, dexamethasone, increased the current and abundance of mature ERG proteins in both hERG-HEK cells and neonatal cardiac myocytes through the enhancement of SGK1 but not SGK3 expression. We have previously shown that mature hERG channels are degraded by ubiquitin ligase Nedd4-2 via enhanced channel ubiquitination. Here, we showed that SGK1 or SGK3 overexpression increased Nedd4-2 phosphorylation, which is known to inhibit Nedd4-2 activity. Nonetheless, disruption of the Nedd4-2 binding site in hERG channels did not eliminate the SGK-induced increase in hERG expression. Additional disruption of Rab11 proteins led to a complete elimination of SGK-mediated increase in hERG expression. These results show that SGK enhances the expression level of mature hERG channels by inhibiting Nedd4-2 as well as by promoting Rab11-mediated hERG recycling.     

Endoplasmic reticulum retention and rescue by heteromeric assembly regulate human ERG 1a/1b surface channel composition

Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in I(Kr)-mediated repolarization. Native I(Kr) channels are composed of two alpha subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an "RXR" ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels.     

hERG1 channel activators: A new anti-arrhythmic principle

The cardiac action potential is the result of an orchestrated function of a number of different ion channels. Action potential repolarisation in humans relies on three potassium current components named I_Kr, I_Ks and I_K1 with party overlapping functions. The ion channel α-subunits conducting these currents are hERG1 (Kv11.1), KCNQ1 (Kv7.1) and Kir2.1. Loss-of-function in any of these currents can result in long QT syndrome. Long QT is a pro-arrhythmic disease with increased risk of developing lethal ventricular arrhythmias such as Torsade de Pointes and ventricular fibrillation. In addition to congenital long QT, acquired long QT can also constitute a safety risk. Especially unintended inhibition of the hERG1 channel constitutes a major concern in the development of new drugs. Based on this knowledge is has been speculated whether activation of the hERG1 channel could be anti-arrhythmic and thereby constitute a new principle in treatment of cardiac arrhythmogenic disorders. The first hERG1 channel agonist was reported in 2005 and a limited number of such compounds are now available. In the present text we review results obtained by hERG1 channel activation in a number of cardiac relevant settings from in vitro to in vivo. It is demonstrated how the principle of hERG1 channel activation under certain circumstances can constitute a new anti-arrhythmogenic principle. Finally, important conceptual differences between the short QT syndrome and the hERG1 channel activation, are evaluated.     

hERG1a/1b heteromeric currents exhibit amplified attenuation of inactivation in variant 1 short QT syndrome

Potassium channels encoded by hERG (human ether-à-go-go-related gene) underlie the cardiac rapid delayed rectifier K⁺ current (I_Kr) and hERG mutations underpin clinically important repolarization disorders. Virtually all electrophysiological investigations of hERG mutations have studied exclusively the hERG1a isoform; however, recent evidence indicates that native I_Kr channels may be comprised of hERG1a together with the hERG1b variant, which has a shorter N-terminus. Here, for the first time, electrophysiological effects were studied of a gain-of-function hERG mutation (N588K; responsible for the ‘SQT1’ variant of the short QT syndrome) on current (I_hERG1a/1b) carried by co-expressed hERG1a/1b channels. There were no significant effects of N588K on I_hERG1a/1b activation or deactivation, but N588K I_hERG1a/1b showed little inactivation up to highly positive voltages (?+80 mV), a more marked effect than seen for hERG1a expressed alone. I_hERG1a/1b under action potential voltage-clamp, and the effects on this of the N588K mutation, also showed differences from those previously reported for hERG1a. The amplified attenuation of I_hERG inactivation for the N588K mutation reported here indicates that the study of co-expressed hERG1a/1b channels should be considered when investigating clinically relevant hERG channel mutations, even if these reside outside of the N-terminus region.     

26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

Mutations of the cyclic nucleotide binding domain (CNBD) may disrupt human ether-a-go-go-related gene (hERG) K(+) channel function and lead to hereditary long QT syndrome (LQTS). We identified a novel missense mutation located in close proximity to the CNBD, hERG R744P, in a patient presenting with recurrent syncope and aborted cardiac death triggered by sudden auditory stimuli. Functional properties of wild type (WT) and mutant hERG R744P subunits were studied in Xenopus laevis oocytes using two-electrode voltage clamp electrophysiology and Western blot analysis. HERG R744P channels exhibited reduced activating currents compared to hERG WT (1.48±0.26 versus 3.40±0.29μA; n=40). These findings were confirmed by tail current analysis (hERG R744P, 0.53±0.07μA; hERG WT, 0.97±0.06μA; n=40). Cell surface trafficking of hERG R744P protein subunits was not impaired. To simulate the autosomal-dominant inheritance associated with LQTS, WT and R744P subunits were co-expressed in equimolar ratio. Mean activating and tail currents were reduced by 32% and 25% compared to hERG WT (n=40), indicating that R744P protein did not exert dominant-negative effects on WT channels. The half-maximal activation voltage was not significantly affected by the R744P mutation. This study highlights the significance of in vitro testing to provide mechanistic evidence for pathogenicity of mutations identified in LQTS. The functional defect associated with hERG R744P serves as molecular basis for LQTS in the index patient.     

Isoform-Specific Dominant-Negative Effects Associated with hERG1 G628S Mutation in Long QT Syndrome

Background

Mutations in the human ether-a-go-go-related gene 1 (hERG1) cause type 2 long QT syndrome (LQT2). The hERG1 gene encodes a K⁺ channel with properties similar to the rapidly activating delayed rectifying K⁺ current in the heart. Several hERG1 isoforms with unique structural and functional properties have been identified. To date, the pathogenic mechanisms of LQT2 mutations have been predominantly described in the context of the hERG1a isoform. In the present study, we investigated the functional consequences of the LQT2 mutation G628S in the hERG1b and hERG1a_USO isoforms.

Methods

A double-stable, mammalian expression system was developed to characterize isoform-specific dominant-negative effects of G628S-containing channels when co-expressed at equivalent levels with wild-type hERG1a. Western blot and co-immunoprecipitation studies were performed to study the trafficking and co-assembly of wild-type and mutant hERG1 isoforms. Patch-clamp electrophysiology was performed to characterize hERG1 channel function and the isoform-specific dominant-negative effects associated with the G628S mutation.

Conclusions

The non-functional hERG1a-G628S and hERG1b-G628S channels co-assembled with wild-type hERG1a and dominantly suppressed hERG1 current. In contrast, G628S-induced dominant-negative effects were absent in the context of the hERG1a_USO isoform. hERG1a_USO-G628S channels did not appreciably associate with hERG1a and did not significantly suppress hERG1 current when co-expressed at equivalent ratios or at ratios that approximate those found in cardiac tissue. These results suggest that the dominant-negative effects of LQT2 mutations may primarily occur in the context of the hERG1a and hERG1b isoforms.     

MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia

A novel potassium channel gene has been cloned, characterized, and associated with cardiac arrhythmia. The gene encodes MinK-related peptide 1 (MiRP1), a small integral membrane subunit that assembles with HERG, a pore-forming protein, to alter its function. Unlike channels formed only with HERG, mixed complexes resemble native cardiac IKr channels in their gating, unitary conductance, regulation by potassium, and distinctive biphasic inhibition by the class III antiarrhythmic E-4031. Three missense mutations associated with long QT syndrome and ventricular fibrillation are identified in the gene for MiRP1. Mutants form channels that open slowly and close rapidly, thereby diminishing potassium currents. One variant, associated with clarithromycin-induced arrhythmia, increases channel blockade by the antibiotic. A mechanism for acquired arrhythmia is revealed: genetically based reduction in potassium currents that remains clinically silent until combined with additional stressors.     

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.     

Rescue of aberrant gating by a genetically encoded PAS (Per-Arnt-Sim) domain in several long QT syndrome mutant human ether-á-go-go-related gene potassium channels

Congenital long QT syndrome 2 (LQT2) is caused by loss-of-function mutations in the human ether-á-go-go-related gene (hERG) voltage-gated potassium (K(+)) channel. hERG channels have slow deactivation kinetics that are regulated by an N-terminal Per-Arnt-Sim (PAS) domain. Only a small percentage of hERG channels containing PAS domain LQT2 mutations (hERG PAS-LQT2) have been characterized in mammalian cells, so the functional effect of these mutations is unclear. We investigated 11 hERG PAS-LQT2 channels in HEK293 cells and report a diversity of functional defects. Most hERG PAS-LQT2 channels formed functional channels at the plasma membrane, as measured by whole cell patch clamp recordings and cell surface biotinylation. Mutations located on one face of the PAS domain (K28E, F29L, N33T, R56Q, and M124R) caused defective channel gating, including faster deactivation kinetics and less steady-state inactivation. Conversely, the other mutations caused no measurable differences in channel gating (G53R, H70R, and A78P) or no measurable currents (Y43C, C66G, and L86R). We used a genetically encoded hERG PAS domain (NPAS) to examine whether channel dysfunction could be corrected. We found that NPAS fully restored wild-type-like deactivation kinetics and steady-state inactivation to the hERG PAS-LQT2 channels. Additionally, NPAS rescued aberrant currents in hERG R56Q channels during a dynamic ramp voltage clamp. Thus, our results reveal a putative "gating face" in the PAS domain where mutations within this region form functional channels with altered gating properties, and we show that NPAS is a general means for rescuing aberrant gating in hERG LQT2 mutant channels and may be a potential biological therapeutic.     

Endocytosis of hERG Is Clathrin-Independent and Involves Arf6

The hERG potassium channel is critical for repolarisation of the cardiac action potential. Reduced expression of hERG at the plasma membrane, whether caused by hereditary mutations or drugs, results in long QT syndrome and increases the risk of ventricular arrhythmias. Thus, it is of fundamental importance to understand how the density of this channel at the plasma membrane is regulated. We used antibodies to an extracellular native or engineered epitope, in conjunction with immunofluorescence and ELISA, to investigate the mechanism of hERG endocytosis in recombinant cells and validated the findings in rat neonatal cardiac myocytes. The data reveal that this channel undergoes rapid internalisation, which is inhibited by neither dynasore, an inhibitor of dynamin, nor a dominant negative construct of Rab5a, into endosomes that are largely devoid of the transferrin receptor. These results support a clathrin-independent mechanism of endocytosis and exclude involvement of dynamin-dependent caveolin and RhoA mechanisms. In agreement, internalised hERG displayed marked overlap with glycosylphosphatidylinositol-anchored GFP, a clathrin-independent cargo. Endocytosis was significantly affected by cholesterol extraction with methyl-β-cyclodextrin and inhibition of Arf6 function with dominant negative Arf6-T27N-eGFP. Taken together, we conclude that hERG undergoes clathrin-independent endocytosis via a mechanism involving Arf6.     

Cell Surface Expression of Human Ether-a-go-go-related Gene (hERG) Channels Is Regulated by Caveolin-3 Protein via the Ubiquitin Ligase Nedd4-2

The human ether-a-go-go-related gene (hERG) encodes the rapidly activating delayed rectifier potassium channel (I_Kr) which plays an important role in cardiac repolarization. A reduction or increase in hERG current can cause long or short QT syndrome, respectively, leading to fatal cardiac arrhythmias. The channel density in the plasma membrane is a key determinant of the whole cell current amplitude. To gain insight into the molecular mechanisms for the regulation of hERG density at the plasma membrane, we used whole cell voltage clamp, Western blotting, and immunocytochemical methods to investigate the effects of an integral membrane protein, caveolin-3 (Cav3) on hERG expression levels. Our data demonstrate that Cav3, hERG, and ubiquitin-ligase Nedd4-2 interact with each other and form a complex. Expression of Cav3 thus enhances the hERG-Nedd4-2 interaction, leading to an increased ubiquitination and degradation of mature, plasma-membrane localized hERG channels. Disrupting Nedd4-2 interaction with hERG by mutations eliminates the effects of Cav3 on hERG channels. Knockdown of endogenous Cav3 or Nedd4-2 in cultured neonatal rat ventricular myocytes using siRNA led to an increase in native I_Kr. Our data demonstrate that hERG expression in the plasma membrane is regulated by Cav3 via Nedd4-2. These findings extend our understanding of the regulation of hERG channels and cardiac electrophysiology.     

Expression of distinct ERG proteins in rat, mouse, and human heart. Relation to functional I(Kr) channels

One form of inherited long QT syndrome, LQT2, results from mutations in HERG1, the human ether-a-go-go-related gene, which encodes a voltage-gated K(+) channel alpha subunit. Heterologous expression of HERG1 gives rise to K(+) currents that are similar (but not identical) to the rapid component of delayed rectification, I(Kr), in cardiac myocytes. In addition, N-terminal splice variants of HERG1 and MERG1 (mouse ERG1) referred to as HERG1b and MERG1b have been cloned and suggested to play roles in the generation of functional I(Kr) channels. In the experiments here, antibodies generated against HERG1 were used to examine ERG1 protein expression in heart and in brain. In Western blots of extracts of QT-6 cells expressing HERG1, MERG1, or RERG1 (rat ERG1) probed with antibodies targeted against the C terminus of HERG1, a single 155-kDa protein is identified, whereas a 95-kDa band is evident in blots of extracts from cells expressing MERG1b or HERG1b. In immunoblots of fractionated rat (and mouse) brain and heart membrane proteins, however, two prominent high molecular mass proteins of 165 and 205 kDa were detected. Following treatment with glycopeptidase F, the 165- and 205-kDa proteins were replaced by two new bands at 175 and 130 kDa, suggesting that ERG1 is differentially glycosylated in rat/mouse brain and heart. In human heart, a single HERG1 protein with an apparent molecular mass of 145 kDa is evident. In rats, ERG1 protein (and I(Kr)) expression is higher in atria than ventricles, whereas in humans, HERG1 expression is higher in ventricular, than atrial, tissue. Taken together, these results suggest that the N-terminal alternatively spliced variants of ERG1 (i.e. ERG1b) are not expressed at the protein level in rat, mouse, or human heart and that these variants do not, therefore, play roles in the generation of functional cardiac I(Kr) channels.     

Stoichiometry of altered hERG1 channel gating by small molecule activators

Voltage-gated K⁺ channels are tetramers formed by coassembly of four identical or highly related subunits. All four subunits contribute to formation of the selectivity filter, the narrowest region of the channel pore which determines K⁺ selective conductance. In some K⁺ channels, the selectivity filter can undergo a conformational change to reduce K⁺ flux by a mechanism called C-type inactivation. In human ether-a-go-go–related gene 1 (hERG1) K⁺ channels, C-type inactivation is allosterically inhibited by ICA-105574, a substituted benzamide. PD-118057, a 2-(phenylamino) benzoic acid, alters selectivity filter gating to enhance open probability of channels. Both compounds bind to a hydrophobic pocket located between adjacent hERG1 subunits. Accordingly, a homotetrameric channel contains four identical activator binding sites. Here we determine the number of binding sites required for maximal drug effect and determine the role of subunit interactions in the modulation of hERG1 gating by these compounds. Concatenated tetramers were constructed to contain a variable number (zero to four) of wild-type and mutant hERG1 subunits, either L646E to inhibit PD-118057 binding or F557L to inhibit ICA-105574 binding. Enhancement of hERG1 channel current magnitude by PD-118057 and attenuated inactivation by ICA-105574 were mediated by cooperative subunit interactions. Maximal effects of the both compounds required the presence of all four binding sites. Understanding how hERG1 agonists allosterically modify channel gating may facilitate mechanism-based drug design of novel agents for treatment of long QT syndrome.     

A place for high-throughput electrophysiology in cardiac safety: screening hERG cell lines and novel compounds with the ion works HTTM system

Several commercially available pharmaceutical compounds have been shown to block the IKr current of the cardiac action potential. This effect can cause a prolongation of the electrocardiogram QT interval and a delay in ventricular repolarization. The Food and Drug Administration recommends that all new potential drug candidates be assessed for IKr block to avoid a potentially lethal cardiac arrhythmia known as torsades de pointes. Direct compound interaction with the human ether-a-go-go- related gene (hERG) product, a delayed rectifier potassium channel, has been identified as a molecular mechanism of IKr block. One strategy to identify compounds with hERG liability is to monitor hERG current inhibition using electrophysiology techniques. The authors describe the Ion Works HT instrument as a tool for screening cell lines expressing hERG channels. Based on current amplitude and stability criteria, a cell line was selected and used to perform a 300-compound screen. The screen was able to identify compounds with hERG activity within projects that spanned different therapeutic areas. The cell line selection and optimization, as well as the screening abilities of the Ion Works HT system, provide a powerful means of assessing hERGactive compounds early in the drug discovery pipeline.     

Human ether-a-go-go related gene (hERG) K channels: Function and dysfunction

The human Ether-a-go-go Related Gene (hERG) potassium channel plays a central role in regulating cardiac excitability and maintenance of normal cardiac rhythm. Mutations in hERG cause a third of all cases of congenital long QT syndrome, a disorder of cardiac repolarisation characterised by prolongation of the QT interval on the surface electrocardiogram, abnormal T waves, and a risk of sudden cardiac death due to ventricular arrhythmias. Additionally, the hERG channel protein is the molecular target for almost all drugs that cause the acquired form of long QT syndrome. Advances in understanding the structural basis of hERG gating, its traffic to the cell surface, and the molecular architecture involved in drug-block of hERG, are providing the foundation for rational treatment and prevention of hERG associated long QT syndrome. This review summarises the current knowledge of hERG function and dysfunction, and the areas of ongoing research.     

Block of the human ether-a-go-go-related gene (hERG) K channel by the antidepressant desipramine

Desipramine is a tricyclic antidepressant for psychiatric disorders that can induce QT prolongation, which may lead to torsades de pointes. Since blockade of cardiac human ether-a-go-go-related gene (hERG) channels is an important cause of acquired long QT syndrome, we investigated the acute effects of desipramine on hERG channels to determine the electrophysiological basis for its pro-arrhythmic potential. We examined the effects of desipramine on the hERG channels expressed in Xenopus oocytes using two-microelectrode voltage-clamp techniques. Desipramine-induced concentration-dependent decreases in the current amplitude at the end of the voltage steps and hERG tail currents. The IC₅₀ for desipramine needed to block the hERG current in Xenopus oocytes decreased progressively relative to the degree of depolarization. Desipramine affected the channels in the activated and inactivated states but not in the closed states. The S6 domain mutations, Tyr-652 located in the S6 domain of the hERG channel reduced the potency of the channel block by desipramine more than a mutation of Phe-656 in the same region. These results suggest that desipramine is a blocker of the hERG channels, providing a molecular mechanism for the arrhythmogenic side effects during the clinical administration of desipramine.     

Involvement of Caveolin in Low K+-induced Endocytic Degradation of Cell-surface Human Ether-a-go-go-related Gene (hERG) Channels

Reduction in the rapidly activating delayed rectifier K⁺ channel current (I_Kr) due to either mutations in the human ether-a-go-go-related gene (hERG) or drug block causes inherited or drug-induced long QT syndrome. A reduction in extracellular K⁺ concentration ([K⁺]_o) exacerbates long QT syndrome. Recently, we demonstrated that lowering [K⁺]_o promotes degradation of I_Kr in rabbit ventricular myocytes and of the hERG channel stably expressed in HEK 293 cells. In this study, we investigated the degradation pathways of hERG channels under low K⁺ conditions. We demonstrate that under low K⁺ conditions, mature hERG channels and caveolin-1 (Cav1) displayed a parallel time-dependent reduction. Mature hERG channels coprecipitated with Cav1 in co-immunoprecipitation analysis, and internalized hERG channels colocalized with Cav1 in immunocytochemistry analysis. Overexpression of Cav1 accelerated internalization of mature hERG channels in 0 mm K⁺_o, whereas knockdown of Cav1 impeded this process. In addition, knockdown of dynamin 2 using siRNA transfection significantly impeded hERG internalization and degradation under low K⁺_o conditions. In cultured neonatal rat ventricular myocytes, knockdown of caveolin-3 significantly impeded low K⁺_o-induced reduction of I_Kr. Our data indicate that a caveolin-dependent endocytic route is involved in low K⁺_o-induced degradation of mature hERG channels.