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.     

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit of the rapidly activating delayed rectifier K⁺ channel in the heart, which plays a critical role in cardiac action potential repolarization. Dysfunction of I_Kr causes long QT syndrome, a cardiac electrical disorder that predisposes affected individuals to fatal arrhythmias and sudden death. The homeostasis of hERG channels in the plasma membrane depends on a balance between protein synthesis and degradation. Our recent data indicate that hERG channels undergo enhanced endocytic degradation under low potassium (hypokalemia) conditions. The GTPase Rab4 is known to mediate rapid recycling of various internalized proteins to the plasma membrane. In the present study, we investigated the effect of Rab4 on the expression level of hERG channels. Our data revealed that overexpression of Rab4 decreases the expression level of hERG in the plasma membrane. Rab4 does not affect the expression level of the Kv1.5 or EAG K⁺ channels. Mechanistically, our data demonstrate that overexpression of Rab4 increases the expression level of endogenous Nedd4-2, a ubiquitin ligase that targets hERG but not Kv1.5 or EAG channels for ubiquitination and degradation. Nedd4-2 undergoes self- ubiquitination and degradation. Rab4 interferes with Nedd4-2 degradation, resulting in an increased expression level of Nedd4-2, which targets hERG. In summary, the present study demonstrates a novel pathway for hERG regulation; Rab4 decreases the hERG density at the plasma membrane by increasing the endogenous Nedd4-2 expression.     

Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel

The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (I_Kr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K⁺ conditions. In the present study, we addressed whether hERG internalization occurs under normal K⁺ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K⁺-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K⁺ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K⁺ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels.     

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.     

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 IC₇₀ 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 IC₅₀ 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.     

Enhanced inhibitory effect of acidosis on hERG potassium channels that incorporate the hERG1b isoform

Extracellular acidosis occurs in the heart during myocardial ischemia and can lead to dangerous arrhythmias. Potassium channels encoded by hERG (human ether-à-go-go-related gene) mediate the cardiac rapid delayed rectifier K⁺ current (I_Kr), and impaired hERG function can exacerbate arrhythmia risk. Nearly all electrophysiological investigations of hERG have centred on the hERG1a isoform, although native I_Kr channels may be comprised of hERG1a and hERG1b, which has a unique shorter N-terminus. This study has characterised for the first time the effects of extracellular acidosis (an extracellular pH decrease from 7.4 to 6.3) on hERG channels incorporating the hERG1b isoform. Acidosis inhibited hERG1b current amplitude to a significantly greater extent than that of hERG1a, with intermediate effects on coexpressed hERG1a/1b. I_hERG tail deactivation was accelerated by acidosis for both isoforms. hERG1a/1b activation was positively voltage-shifted by acidosis, and the fully-activated current–voltage relation was reduced in amplitude and right-shifted (by ∼10 mV). Peak I_hERG1a/1b during both ventricular and atrial action potentials was both suppressed and positively voltage-shifted by acidosis. Differential expression of hERG isoforms may contribute to regional differences in I_Kr in the heart. Therefore inhibitory effects of acidosis on I_Kr could also differ regionally, depending on the relative expression levels of hERG1a and 1b, thereby increasing dispersion of repolarization and arrhythmia risk.     

Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification

Two-pore domain K⁺ channels (K2P) mediate background K⁺ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K⁺ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH_o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH_o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na⁺-based bath solutions with physiological K⁺ gradients, lowered pH_o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K⁺ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na⁺ to K⁺ relative permeability. Low pH_o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na⁺-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na⁺ to K⁺ relative permeability and decreases in electrochemical driving forces of outward K⁺ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K⁺ channels change ion selectivity in response to lowered pH_o, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.     

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.     

The Extracellular K+ Concentration Dependence of Outward Currents through Kir2.1 Channels Is Regulated by Extracellular Na+ and Ca2+

It has been known for more than three decades that outward Kir currents (I_K1) increase with increasing extracellular K⁺ concentration ([K⁺]_o). Although this increase in I_K1 can have significant impacts under pathophysiological cardiac conditions, where [K⁺]_o can be as high as 18 mm and thus predispose the heart to re-entrant ventricular arrhythmias, the underlying mechanism has remained unclear. Here, we show that the steep [K⁺]_o dependence of Kir2.1-mediated outward I_K1 was due to [K⁺]_o-dependent inhibition of outward I_K1 by extracellular Na⁺ and Ca²⁺. This could be accounted for by Na⁺/Ca²⁺ inhibition of I_K1 through screening of local negative surface charges. Consistent with this, extracellular Na⁺ and Ca²⁺ reduced the outward single-channel current and did not increase open-state noise or decrease the mean open time. In addition, neutralizing negative surface charges with a carboxylate esterifying agent inhibited outward I_K1 in a similar [K⁺]_o-dependent manner as Na⁺/Ca²⁺. Site-directed mutagenesis studies identified Asp¹¹⁴ and Glu¹⁵³ as the source of surface charges. Reducing K⁺ activation and surface electrostatic effects in an R148Y mutant mimicked the action of extracellular Na⁺ and Ca²⁺, suggesting that in addition to exerting a surface electrostatic effect, Na⁺ and Ca²⁺ might inhibit outward I_K1 by inhibiting K⁺ activation. This study identified interactions of K⁺ with Na⁺ and Ca²⁺ that are important for the [K⁺]_o dependence of Kir2.1-mediated outward I_K1.     

Small GTPase Determinants for the Golgi Processing and Plasmalemmal Expression of Human Ether-a-go-go Related (hERG) K+ Channels

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1–10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K⁺ channel α-subunits that form the pore of the rapidly activating delayed rectifier K⁺ current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I_hERG) by 85% (n ≥ 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I_hERG by 79% (n ≥ 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.The human KCNH2 or ether-a-go-go related gene (hERG)³ encodes the voltage-gated K⁺ channel α-subunits that oligomerize to form the pore of the rapidly activating delayed rectifier K⁺ current (I_Kr) in cardiac myocytes (1–3). Hundreds of hERG mutations are linked to the congenital pro-arrhythmic Type 2 Long QT syndrome (LQT2) and functional studies suggest that these mutations result in a loss of normal hERG K⁺ channel (hERG) function (4, 5). In LQT2, missense mutations are the dominant abnormality and many LQT2 missense mutations reduce hERG K⁺ current (I_hERG) by decreasing the intracellular transport or trafficking of hERG to the Golgi apparatus (Golgi) and the cell surface membrane (plasmalemma) (6). Therefore, disruption of hERG K⁺ channel trafficking appears to be a principal mechanism for disease.Movement of proteins between membrane-bound intracellular compartments is mediated by small transport vesicles, which bud from a donor compartment to fuse with an appropriate acceptor compartment. The trafficking of many transmembrane and secretory proteins between the ER and Golgi compartments is dependent on the small GTPases ADP-ribosylation factor 1 (ARF1) and Sar1, which regulate the formation of coat-associated protein complex I (COPI) and II (COPII) vesicles, respectively (7–19). These small GTPases facilitate the polymerization of transport vesicle protein coats on the donor membrane. Vesicular cargo selection, docking, and fusion to the target membrane are regulated by adaptor proteins, SNARE proteins, and Rab GTPases. To rationally develop novel therapeutic targets that may increase the expression of trafficking-deficient LQT2 mutant channels, the molecular mechanisms that regulate the trafficking of hERG need to be explored. The purpose of this study is to identify transport proteins that regulate the trafficking of wild type (WT) hERG. We used a strategy of testing specific WT GTPases or ones containing dominant negative (DN) mutations to interfere with their function.     

The role of monoubiquitination in endocytic degradation of human ether-a-go-go-related gene (hERG) channels under low K+ conditions

A reduction in extracellular K(+) concentration ([K(+)](o)) causes cardiac arrhythmias and triggers internalization of the cardiac rapidly activating delayed rectifier potassium channel (I(Kr)) encoded by the human ether-a-go-go-related gene (hERG). We investigated the role of ubiquitin (Ub) in endocytic degradation of hERG channels stably expressed in HEK cells. Under low K(+) conditions, UbKO, a lysine-less mutant Ub that only supports monoubiquitination, preferentially interacted and selectively enhanced degradation of the mature hERG channels. Overexpression of Vps24 protein, also known as charged multivesicular body protein 3, significantly accelerated degradation of mature hERG channels, whereas knockdown of Vps24 impeded this process. Moreover, the lysosomal inhibitor bafilomycin A1 inhibited degradation of the internalized mature hERG channels. Thus, monoubiquitination directs mature hERG channels to degrade through the multivesicular body/lysosome pathway. Interestingly, the protease inhibitor lactacystin inhibited the low K(+)-induced hERG endocytosis and concomitantly led to an accumulation of monoubiquitinated mature hERG channels, suggesting that deubiquitination is also required for the endocytic degradation. Consistently, overexpression of the endosomal deubiquitinating enzyme signal transducing adaptor molecule-binding protein significantly accelerated whereas knockdown of endogenous signal transducing adaptor molecule-binding protein impeded degradation of the mature hERG channels under low K(+) conditions. Thus, monoubiquitin dynamically mediates endocytic degradation of mature hERG channels under low K(+) conditions.     

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

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.     

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.     

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.     

Quantitative prediction of the arrhythmogenic effects of de novo hERG mutations in computational models of human ventricular tissues

Mutations to hERG which result in changes to the rapid delayed rectifier current I _Kr can cause long and short QT syndromes and are associated with an increased risk of cardiac arrhythmias. Experimental recordings of I _Kr reveal the effects of mutations at the channel level, but how these changes translate to the cell and tissue levels remains unclear. We used computational models of human ventricular myocytes and tissues to predict and quantify the effects that de novo hERG mutations would have on cell and tissue electrophysiology. Mutations that decreased I _Kr maximum conductance resulted in an increased cell and tissue action potential duration (APD) and a long QT interval on the electrocardiogram (ECG), whereas those that caused a positive shift in the inactivation curve resulted in a decreased APD and a short QT. Tissue vulnerability to re-entrant arrhythmias was correlated with transmural dispersion of repolarisation, and any change to this vulnerability could be inferred from the ECG QT interval or T wave peak-to-end time. Faster I _Kr activation kinetics caused cell APD alternans to appear over a wider range of pacing rates and with a larger magnitude, and spatial heterogeneity in these cellular alternans resulted in discordant alternans at the tissue level. Thus, from channel kinetic data, we can predict the tissue-level electrophysiological effects of any hERG mutations and identify how the mutation would manifest clinically, as either a long or short QT syndrome with or without an increased risk of alternans and re-entrant arrhythmias.     

Epigallocatechin-3-gallate elicits Ca spike in MCF-7 breast cancer cells: Essential role of Cav3.2 channels

We used MCF-7 human breast cancer cells that endogenously express Cav3.1 and Cav3.2 T-type Ca²⁺ channels toward a mechanistic study on the effect of EGCG on [Ca²⁺]_i. Confocal Ca²⁺ imaging showed that EGCG induces a [Ca²⁺]_i spike which is due to extracellular Ca²⁺ entry and is sensitive to catalase and to low-specificity (mibefradil) and high-specificity (Z944) T-type Ca²⁺channel blockers. siRNA knockdown of T-type Ca²⁺ channels indicated the involvement of Cav3.2 but not Cav3.1. Application of EGCG to HEK cells expressing either Cav3.2 or Cav3.1 induced enhancement of Cav3.2 and inhibition of Cav3.1 channel activity. Measurements of K⁺ currents in MCF-7 cells showed a reversible, catalase-sensitive inhibitory effect of EGCG, while siRNA for the Kv1.1 K⁺ channel induced a reduction of the EGCG [Ca²⁺]_i spike. siRNA for Cav3.2 reduced EGCG cytotoxicity to MCF-7 cells, as measured by calcein viability assay. Together, data suggest that EGCG promotes the activation of Cav3.2 channels through K⁺ current inhibition leading to membrane depolarization, and in addition increases Cav3.2 currents. Cav3.2 channels are in part responsible for EGCG inhibition of MCF-7 viability, suggesting that deregulation of [Ca²⁺]_i by EGCG may be relevant in breast cancer treatment.     

Potassium Channel Modulation by a Toxin Domain in Matrix Metalloprotease 23

Peptide toxins found in a wide array of venoms block K⁺ channels, causing profound physiological and pathological effects. Here we describe the first functional K⁺ channel-blocking toxin domain in a mammalian protein. MMP23 (matrix metalloprotease 23) contains a domain (MMP23_TxD) that is evolutionarily related to peptide toxins from sea anemones. MMP23_TxD shows close structural similarity to the sea anemone toxins BgK and ShK. Moreover, this domain blocks K⁺ channels in the nanomolar to low micromolar range (Kv1.6 > Kv1.3 > Kv1.1 = Kv3.2 > Kv1.4, in decreasing order of potency) while sparing other K⁺ channels (Kv1.2, Kv1.5, Kv1.7, and KCa3.1). Full-length MMP23 suppresses K⁺ channels by co-localizing with and trapping MMP23_TxD-sensitive channels in the ER. Our results provide clues to the structure and function of the vast family of proteins that contain domains related to sea anemone toxins. Evolutionary pressure to maintain a channel-modulatory function may contribute to the conservation of this domain throughout the plant and animal kingdoms.     

The role of hERG1 K channels and a functional link between hERG1 K channels and SDF-1 in acute leukemic cell migration

Stromal cell-derived factor-1 (SDF-1) and its unique receptor, CXCR4, regulate stem/progenitor cell migration and retention in the bone marrow and are required for hematopoiesis. Recent studies found that hERG1 K⁺ channels were important regulators of tumor cell migration. In this study, we investigated whether SDF-1 induced acute leukemic cell migration associated with hERG1 K⁺ channels. Our results showed that E-4031, a specific hERG1 K⁺ channels inhibitor, significantly blocked SDF-1-induced migration of leukemic cell lines, primary acute leukemic cells, leukemic stem cells and HEK293T cells transfected with herg-pEGFP. The migration of phenotypically recognizable subsets gave the indication that lymphoblastic leukemic cells were inhibited more than myeloid cells while in the presence of E-4031 which maybe associated with herg expression. SDF-1 increased hERG1 K⁺ current expressed in oocytes and HEK293T cells transfected with herg-pEGFP. There were no significant changes of CXCR4 expression on both HL-60 cells and primary leukemic cells regardless if untreated or treated with E-4031 for 24 h (P > 0.05). The hERG1 K⁺ current increased by SDF-1 might contribute to the mechanism of SDF-1-induced leukemic cell migration. The data suggested that hERG1 K⁺ channels functionally linked to cell migration induced by SDF-1.     

Structure and function of potassium channels in plants: some inferences about the molecular origin of inward rectification in KAT1 channels (Review)

Potassium channels in plants play a variety of important physiological roles including K⁺ uptake into roots, stomatal and leaf movements, and release of K⁺ into the xylem. This review summarizes current knowledge about a class of plant genes whose products are K⁺ channel-forming proteins. Potassium channels of this class belong to a superfamily characterized by six membrane-spanning domains (S1-6), a positively charged S4 domain and a region between the S5 and S6 segments that forms the channel selectivity filter. These channels are voltage dependent, which means the membrane potential modifies the probability of opening (P_o). However, despite these channels sharing the same topology as the outward-rectifying K⁺ channels, which are activated by membrane depolarization, some plant K⁺ channels such as KAT1/2 and KST1 open with hyperpolarizing voltages. In outward-rectifying K⁺ channels, the change in P_o is achieved through a voltage sensor formed by the S4 segment that detects the voltage transferring its energy to the gate that controls pore opening. This coupling is achieved by an outward displacement of the charges contained in S4. In KAT1, most of the results indicate that S4 is the voltage sensor. However, how the movement of S4 leads to opening remains unanswered. On the basis of recent data, we propose here that in plant-inward rectifiers an inward movement of S4 leads to channel opening and that the difference between it and outward-rectifying channels resides in the mechanism that couples gating charge displacement with pore opening.