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Potassium channels stabilize the resting potential and neuronal excitability. Among them, erg (ether-à-go-go-related gene) K + channels represent a subfamily of voltage-gated channels, consisting of erg1, erg2, and erg3 subunits; however, their subunit-specific neuronal functions in vivo are barely understood. To find erg3- and erg1-mediated functions, we generated global Kcnh7 ( erg3 ) and conditional Kcnh2 ( erg1 ) knockout mice. We found that erg3 channels stabilize the resting potential and dampen spontaneous activity in cerebellar Purkinje cells (PCs) and hippocampal CA1 neurons, whereas erg1 channels have suprathreshold functions. Lack of erg3 subunits induced hyperexcitability with increased action potential firing in PCs, but not in CA1 neurons. Notably, erg3 depletion caused depressive-like behavior with reduced locomotor activity, strongly decreased digging behavior, and shorter latencies to fall off a rotating wheel, while learning and memory remained unchanged. Our data show that erg K + channels containing erg3 subunits mediate a neuronal subthreshold K + current that plays important roles in the regulation of locomotor behavior in vivo.

The KCNH family of potassium channels serves relevant physiological functions in both excitable and non-excitable cells, reflected in the massive consequences of mutations or pharmacological manipulation of their function. This group of channels shares structural homology with other voltage-gated K + channels, but the mechanisms of gating in this family show significant differences with respect to the canonical electromechanical coupling in these molecules. In particular, the large intracellular domains of KCNH channels play a crucial role in gating that is still only partly understood. Using KCNH1 (K V 10.1) as a model, we have characterized the behavior of a series of modified channels that could not be explained by the current models. With electrophysiological and biochemical methods combined with mathematical modeling, we show that the uncovering of an open state can explain the behavior of the mutants. This open state, which is not detectable in wild-type channels, appears to lack the rapid flicker block of the conventional open state. Because it is accessed from deep closed states, it elucidates intermediate gating events well ahead of channel opening in the wild type. This allowed us to study gating steps prior to opening, which, for example, explain the mechanism of gating inhibition by Ca 2+ -Calmodulin and generate a model that describes the characteristic features of KCNH channels gating., Competing Interests: RA, AT, AN, LP No competing interests declared, (© 2023, Abdelaziz et al.)

Gliomas are highly malignant brain tumours that remain refractory to treatment. Treatment is typically surgical intervention followed by concomitant temozolomide and radiotherapy; however patient prognosis remains poor. Voltage gated ion channels have emerged as novel targets in cancer therapy and inhibition of a potassium selective subtype (hERG, Kv11.1) has demonstrated antitumour activity. Unfortunately blockade of hERG has been limited by cardiotoxicity, however hERG channel agonists have produced similar chemotherapeutic benefit without significant side effects. In this study, electrophysiological recordings suggest the presence of hERG channels in the anaplastic astrocytoma cell line SMA-560, and treatment with the hERG channel agonist NS1643, resulted in a significant reduction in the proliferation of SMA-560 cells. In addition, NS1643 treatment also resulted in a reduction of the secretion of matrix metalloproteinase-9 and SMA-560 cell migration. When combined with temozolomide, an additive impact was observed, suggesting that NS1643 may be a suitable adjuvant to temozolomide and limit the invasiveness of glioma., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Benn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The main risk factor for cervical cancer is the persistent infection of high-risk HPV subtypes, notably HPV16. Another contributing factor is proinflammatory prostaglandin E 2 (PGE 2 ), a lipid abundantly found in seminal fluid. PGE 2 , along with its receptors (PTGER1-4), contributes to cancer development; however, its specific role in the proliferation of cervical cancer models with high HPV16 copy numbers remains unclear. In this study, we investigated the effects of PGE 2 on the proliferation of CaSki cells, a cell line with a high HPV16 viral load. Surprisingly, PGE 2 inhibited CaSki cell proliferation, while it increased the proliferation of SiHa, HeLa, and C-33 A cervical cancer cells. The effect of PGE 2 on CaSki cell proliferation was specific, as estradiol increased cell growth. Furthermore, PGE 2 suppressed expression and promoter activity of the cervical tumoral marker KCNH1. To discern the specific role of each receptor in cell proliferation, we generated stable CaSki cell lines overexpressing each receptor alongside control cells with an empty vector. Notably, PGE 2 significantly inhibited cell proliferation in all stable transfected CaSki cells, suppressing oncogenic KCNH1 expression and its promoter activity. In conclusion, our findings indicate that PGE 2 inhibits the proliferation of CaSki cervical cancer cells with a high HPV16 load, at least in part, by suppressing the expression of the oncogenic KCNH1 gene., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Drug-induced QT prolongation (diLQT) is a feared side effect that could expose susceptible individuals to fatal arrhythmias. The occurrence of diLQT is primarily attributed to unintended drug interactions with cardiac ion channels, notably the hERG (human ether-a-go-go-related gene) channels that generate the delayed-rectifier potassium current (I Kr ) and thereby regulate the late repolarization phase. There is an important interindividual susceptibility to develop diLQT, which is of unknown origin but can be reproduced in patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs). We aimed to investigate the dynamics of hERG channels in response to sotalol and to identify regulators of the susceptibility to developing diLQT., Methods: We measured electrophysiological activity and cellular distribution of hERG channels after hERG blocker treatment in iPS-CMs derived from patients with highest sensitivity (HS) or lowest sensitivity (LS) to sotalol administration in vivo (ie, on the basis of the measure of the maximal change in QT interval 3 hours after administration). Specific small interfering RNAs and CAVIN1-T2A-GFP adenovirus were used to manipulate CAVIN1 expression., Results: Whereas HS and LS iPS-CMs showed similar electrophysiological characteristics at baseline, the late repolarization phase was prolonged and I Kr significantly decreased after exposure of HS iPS-CMs to low sotalol concentrations. I Kr reduction was caused by a rapid translocation of hERG channel from the membrane to the cytoskeleton-associated fractions upon sotalol application. CAVIN1 , essential for caveolae biogenesis, was 2× more highly expressed in HS iPS-CMs, and its knockdown by small interfering RNA reduced their sensitivity to sotalol. CAVIN1 overexpression in LS iPS-CMs using adenovirus showed reciprocal effects. We found that treatment with sotalol promoted translocation of the hERG channel from the plasma membrane to the cytoskeleton fractions in a process dependent on CAVIN1 (caveolae associated protein 1) expression. CAVIN1 silencing reduced the number of caveolae at the membrane and abrogated the translocation of hERG channel in sotalol-treated HS iPS-CMs. CAVIN1 also controlled cardiomyocyte responses to other hERG blockers, such as E4031, vandetanib, and clarithromycin., Conclusions: Our study identifies unbridled turnover of the potassium channel hERG as a mechanism supporting the interindividual susceptibility underlying diLQT development and demonstrates how this phenomenon is finely tuned by CAVIN1., Competing Interests: Dr Hulot reports research grants from BioSerenity, Sanofi, Servier, and Novo Nordisk, and speaker, advisory board, or consultancy fees from Amgen, AstraZeneca, Bayer, BioSerenity, Bristol Myers Squibb, Novartis, Novo Nordisk, and Vifor Pharma, all unrelated to the present work. The other authors declare no competing financial interests.

The human ether-a-go-go-related gene (hERG) encodes the Kv11.1 (or hERG) channel that conducts the rapidly activating delayed rectifier potassium current (I Kr ). Naturally occurring mutations in hERG impair the channel function and cause long QT syndrome type 2. Many missense hERG mutations lead to a lack of channel expression on the cell surface, representing a major mechanism for the loss-of-function of mutant channels. While it is generally thought that a trafficking defect underlies the lack of channel expression on the cell surface, in the present study, we demonstrate that the trafficking defective mutant hERG G601S can reach the plasma membrane but is unstable and quickly degrades, which is akin to WT hERG channels under low K + conditions. We previously showed that serine (S) residue at 624 in the innermost position of the selectivity filter of hERG is involved in hERG membrane stability such that substitution of serine 624 with threonine (S624T) enhances hERG stability and renders hERG insensitive to low K + culture. Here, we report that the intragenic addition of S624T substitution to trafficking defective hERG mutants G601S, N470D, and P596R led to a complete rescue of the function of these otherwise loss-of-function mutant channels to a level similar to the WT channel, representing the most effective rescue means for the function of mutant hERG channels. These findings not only provide novel insights into hERG mutation-mediated channel dysfunction but also point to the critical role of S624 in hERG stability on the plasma membrane., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Recent clinical studies have highlighted mutations in the voltage-gated potassium channel Kv10.2 encoded by the KCNH5 gene among individuals with autism spectrum disorder (ASD). Our preliminary study found that Kv10.2 was decreased in the hippocampus of valproic acid (VPA) - induced ASD rats. Nevertheless, it is currently unclear how KCNH5 regulates autism-like features, or becomes a new target for autism treatment. We employed KCNH5 knockout (KCNH5 -/- ) rats and VPA - induced ASD rats in this study. Then, we used behavioral assessments, combined with electrophysiological recordings and hippocampal brain slice, to elucidate the impact of KCNH5 deletion and environmental factors on neural development and function in rats. We found that KCNH5 -/- rats showed early developmental delay, neuronal overdevelopment, and abnormal electroencephalogram (EEG) signals, but did not exhibit autism-like behavior. KCNH5 -/- rats exposed to VPA (KCNH5 -/- -VPA) exhibit even more severe autism-like behaviors and abnormal neuronal development. The absence of KCNH5 excessively enhances the activity of the Protein Kinase B (Akt)/Mechanistic Target of Rapamycin (mTOR) signaling pathway in the hippocampus of rats after exposure to VPA. Overall, our findings underscore the deficiency of KCNH5 increases the susceptibility to autism under environmental exposures, suggesting its potential utility as a target for screening and diagnosis in ASD., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Slow deactivation is a critical property of voltage-gated K + channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The voltage-gated potassium ion channel K V 11.1 plays a critical role in cardiac repolarization. Genetic variants that render Kv11.1 dysfunctional cause long QT syndrome (LQTS), which is associated with fatal arrhythmias. Approximately 90% of LQTS-associated variants cause intracellular protein transport (trafficking) dysfunction, which pharmacological chaperones like E-4031 can rescue. Protein folding and trafficking decisions are regulated by chaperones, protein quality control factors, and trafficking machinery comprising the cellular proteostasis network. Here, we test whether trafficking dysfunction is associated with alterations in the proteostasis network of pathogenic Kv11.1 variants and whether pharmacological chaperones can normalize the proteostasis network of responsive variants. We used affinity-purification coupled with tandem mass tag-based quantitative mass spectrometry to assess protein interaction changes of WT K V 11.1 or trafficking-deficient channel variants in the presence or absence of E-4031. We identified 572 core K V 11.1 protein interactors. Trafficking-deficient variants K V 11.1-G601S and K V 11.1-G601S-G965 ∗ had significantly increased interactions with proteins responsible for folding, trafficking, and degradation compared to WT. We confirmed previous findings that the proteasome is critical for K V 11.1 degradation. Our report provides the first comprehensive characterization of protein quality control mechanisms of K V 11.1. We find extensive interactome remodeling associated with trafficking-deficient K V 11.1 variants and with pharmacological chaperone rescue of K V 11.1 cell surface expression. The identified protein interactions could be targeted therapeutically to improve K V 11.1 trafficking and treat LQTS., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Pathogenic variants of the KCNH1 gene can cause dominant-inherited Temple-Baraitser/Zimmermann-Laband syndrome with severe mental retardation, seizure, gingival hyperplasia and nail hypoplasia. This study established an induced pluripotent stem cell (iPSC) line using urinary cells from a girl with KCNH1 recurrent/hotspot pathogenic variant c.1070G > A (p.R357Q). The cell identity, pluripotency, karyotypic integrity, absence of reprogramming virus and mycoplasma contamination, and differential potential to three germ layers of the iPSC line, named as ZJUCHi003, were characterized and confirmed. Furthermore, ZJUCHi003-derived neurons manifested slower action potential repolarization process and wider action potential half-width than the normal neurons. This cell line will be useful for investigating the pathogenic mechanisms of KCNH1 variants-associated symptoms, as well as for evaluating novel therapeutic approaches., (© 2024. The Author(s) under exclusive licence to Japan Human Cell Society.)

Background and Purpose: Drug-induced reduction of the rapid delayed rectifier potassium current carried by the human Ether-à-go-go-Related Gene (hERG) channel is associated with increased risk of arrhythmias. Recent updates to drug safety regulatory guidelines attempt to capture each drug's hERG binding mechanism by combining in vitro assays with in silico simulations. In this study, we investigate the impact on in silico proarrhythmic risk predictions due to uncertainty in the hERG binding mechanism and physiological hERG current model., Experimental Approach: Possible pharmacological binding models were designed for the hERG channel to account for known and postulated small molecule binding mechanisms. After selecting a subset of plausible binding models for each compound through calibration to available voltage-clamp electrophysiology data, we assessed their effects, and the effects of different physiological models, on proarrhythmic risk predictions., Key Results: For some compounds, multiple binding mechanisms can explain the same data produced under the safety testing guidelines, which results in different inferred binding rates. This can result in substantial uncertainty in the predicted torsade risk, which often spans more than one risk category. By comparison, we found that the effect of a different hERG physiological current model on risk classification was subtle., Conclusion and Implications: The approach developed in this study assesses the impact of uncertainty in hERG binding mechanisms on predictions of drug-induced proarrhythmic risk. For some compounds, these results imply the need for additional binding data to decrease uncertainty in safety-critical applications., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

In vitro electrophysiological safety studies have become an integral part of the drug development process because, in many instances, compound-induced QT prolongation has been associated with a direct block of human ether-a-go-go-related gene (hERG) potassium channels or their native current, the rapidly activating delayed rectifier potassium current (I Kr ). Therefore, according to the ICH S7B guideline, the in vitro hERG channel patch-clamp assay is commonly used as an early screen to predict the ability of a compound to prolong the QT interval prior to first-in-human testing. The protocols described in this article are designed to assess the effects of acute or long-term exposure to new chemical entities on the amplitude of I Kr in HEK293 cells stably transfected with the hERG channel (whole-cell configuration of the patch-clamp technique). Examples of results obtained with moxifloxacin, terfenadine, arsenic, pentamidine, erythromycin, and sotalol are provided for illustrative purposes. © 2024 Wiley Periodicals LLC. Basic Protocol: Measurement of the acute effects of test items in the hERG channel test Alternate Protocol: Measurement of the long-term effects of test items in the hERG channel test., (© 2024 Wiley Periodicals LLC.)

The Kv11.1 potassium channel encoded by the Kcnh2 gene is crucial in conducting the rapid delayed rectifier K + current in cardiomyocytes. Homozygous mutation in Kcnh2 is embryonically lethal in humans and mice. However, the molecular signaling pathway of intrauterine fetal loss is unclear. The present study generated a Kcnh2 knockout rat based on edited rat embryonic stem cells (rESCs). Kcnh2 knockout was embryonic lethal on day 11.5 of development due to a heart configuration defect. Experiments with human embryonic heart single cells (6.5‑7 weeks post‑conception) suggested that potassium voltage‑gated channel subfamily H member 2 (KCNH2) plays a crucial role in the development of compact cardiomyocytes. By contrast, apoptosis was found to be triggered in the homozygous embryos, which could be attributed to the failure of KCNH2 to form a complex with integrin β1 that was essential for preventing the process of apoptosis via inhibition of forkhead box O3A. Destruction of the KCNH2/integrin β1 complex reduced the phosphorylation level of AKT and deactivated the glycogen synthase kinase 3 β (GSK‑3β)/β‑catenin pathway, which caused early developmental abnormalities in rats. The present work reveals a basic mechanism by which KCNH2 maintains intact embryonic heart development.

The c.453delC (p.Thr152Profs*14) frameshift mutation in KCNH2 is associated with an elevated risk of Long QT syndrome (LQTS) and fatal arrhythmia. Nevertheless, the loss-of-function mechanism underlying this mutation remains unexplored and necessitates an understanding of electrophysiology. To gain insight into the mechanism of the LQT phenotype, we conducted whole-cell patch-clamp and immunoblot assays, utilizing both a heterologous expression system and patient-derived induced pluripotent stem cell-cardiomyocytes (iPSC-CMs) with 453delC-KCNH2. We also explored the site of translational reinitiation by employing LC/MS mass spectrometry. Contrary to the previous assumption of early termination of translation, the findings of this study indicate that the 453delC-KCNH2 leads to an N-terminally truncated hERG channel, a potential from a non-canonical start codon, with diminished expression and reduced current (IhERG). The co-expression with wildtype KCNH2 produced heteromeric hERG channel with mild dominant-negative effect. Additionally, the heterozygote patient-derived iPSC-CMs exhibited prolonged action potential duration and reduced IhERG, which was ameliorated with the use of a hERG activator, PD-118057. The results of our study offer novel insights into the mechanisms involved in congenital LQTS associated with the 453delC mutation of KCNH2. The mutant results in the formation of less functional N-terminal-truncated channels with reduced amount of membrane expression. A hERG activator is capable of correcting abnormalities in both the heterologous expression system and patient-derived iPSC-CMs., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Zebrafish provide a translational model of human cardiac function. Their similar cardiac electrophysiology enables screening of human cardiac repolarization disorders, drug arrhythmogenicity, and novel antiarrhythmic therapeutics. However, while zebrafish cardiac repolarization is driven by delayed rectifier potassium channel current (I Kr ), the relative role of alternate channel transcripts is uncertain. While human ether-a-go-go-related-gene-1a (hERG1a) is the dominant transcript in humans, expression of the functionally distinct alternate transcript, hERG1b, modifies the electrophysiological and pharmacologic I Kr phenotype. Studies of zebrafish I Kr are frequently translated without consideration for the presence and impact of hERG1b in humans. Here, we performed phylogenetic analyses of all available KCNH genes from Actinopterygii (ray-finned fishes). Our findings confirmed zebrafish cardiac zkcnh6a as the paralog of human hERG1a (hKCNH2a), but also revealed evidence of a hERG1b (hKCNH2b)-like N-terminally truncated gene, zkcnh6b, in zebrafish. zkcnh6b is a teleost-specific variant that resulted from the 3R genome duplication. qRT-PCR showed dominant expression of zkcnh6a in zebrafish atrial and ventricular tissue, with low levels of zkcnh6b. Functional evaluation of zkcnh6b in a heterologous system showed no discernable function under the conditions tested, and no influence on zkcnh6a function during the zebrafish ventricular action potential. Our findings provide the first descriptions of the zkcnh6b gene, and show that, unlike in humans, zebrafish cardiac repolarization does not rely upon co-assembly of zERG1a/zERG1b. Given that hERG1b modifies I Kr function and drug binding in humans, our findings highlight the need for consideration when translating hERG variant effects and toxicological screens in zebrafish, which lack a functional hERG1b-equivalent gene., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ether-a-go-go (EAG) channels are key regulators of neuronal excitability and tumorigenesis. EAG channels contain an N-terminal Per-Arnt-Sim (PAS) domain that can regulate currents from EAG channels by binding small molecules. The molecular mechanism of this regulation is not clear. Using surface plasmon resonance and electrophysiology we show that a small molecule ligand imipramine can bind to the PAS domain of EAG1 channels and inhibit EAG1 currents via this binding. We further used a combination of molecular dynamics (MD) simulations, electrophysiology, and mutagenesis to investigate the molecular mechanism of EAG1 current inhibition by imipramine binding to the PAS domain. We found that Tyr71, located at the entrance to the PAS domain cavity, serves as a "gatekeeper" limiting access of imipramine to the cavity. MD simulations indicate that the hydrophobic electrostatic profile of the cavity facilitates imipramine binding and in silico mutations of hydrophobic cavity-lining residues to negatively charged glutamates decreased imipramine binding. Probing the PAS domain cavity-lining residues with site-directed mutagenesis, guided by MD simulations, identified D39 and R84 as residues essential for the EAG1 channel inhibition by imipramine binding to the PAS domain. Taken together, our study identified specific residues in the PAS domain that could increase or decrease EAG1 current inhibition by imipramine binding to the PAS domain. These findings should further the understanding of molecular mechanisms of EAG1 channel regulation by ligands and facilitate the development of therapeutic agents targeting these channels., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Aims: Long QT syndrome type 2 (LQTS2) is associated with inherited variants in the cardiac human ether-à-go-go-related gene (hERG) K+ channel. However, the pathogenicity of hERG channel gene variants is often uncertain. Using CRISPR-Cas9 gene-edited hiPSC-derived cardiomyocytes (hiPSC-CMs), we investigated the pathogenic mechanism underlying the LQTS-associated hERG R56Q variant and its phenotypic rescue by using the Type 1 hERG activator, RPR260243., Methods and Results: The above approaches enable characterization of the unclear causative mechanism of arrhythmia in the R56Q variant (an N-terminal PAS domain mutation that primarily accelerates channel deactivation) and translational investigation of the potential for targeted pharmacologic manipulation of hERG deactivation. Using perforated patch clamp electrophysiology of single hiPSC-CMs, programmed electrical stimulation showed that the hERG R56Q variant does not significantly alter the mean action potential duration (APD90). However, the R56Q variant increases the beat-to-beat variability in APD90 during pacing at constant cycle lengths, enhances the variance of APD90 during rate transitions, and increases the incidence of 2:1 block. During paired S1-S2 stimulations measuring electrical restitution properties, the R56Q variant was also found to increase the variability in rise time and duration of the response to premature stimulations. Application of the hERG channel activator, RPR260243, reduces the APD variance in hERG R56Q hiPSC-CMs, reduces the variability in responses to premature stimulations, and increases the post-repolarization refractoriness., Conclusion: Based on our findings, we propose that the hERG R56Q variant leads to heterogeneous APD dynamics, which could result in spatial dispersion of repolarization and increased risk for re-entry without significantly affecting the average APD90. Furthermore, our data highlight the antiarrhythmic potential of targeted slowing of hERG deactivation gating, which we demonstrate increases protection against premature action potentials and reduces electrical heterogeneity in hiPSC-CMs., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The hERG1 potassium channel is aberrantly over expressed in tumors and regulates the cancer cell response to integrin-dependent adhesion. We unravel a novel signaling pathway by which integrin engagement by the ECM protein fibronectin promotes hERG1 translocation to the plasma membrane and its association with β1 integrins, by activating girdin-dependent Gαi3 proteins and protein kinase B (Akt). By sequestering hERG1, β1 integrins make it avoid Rab5-mediated endocytosis, where unbound channels are degraded. The cycle of hERG1 expression determines the resting potential (V rest ) oscillations and drives the cortical f-actin dynamics and thus cell motility. To interpret the slow biphasic kinetics of hERG1/β1 integrin interplay, we developed a mathematical model based on a generic balanced inactivation-like module. Integrin-mediated cell adhesion triggers two contrary responses: a rapid stimulation of hERG1/β1 complex formation, followed by a slow inhibition which restores the initial condition. The protracted hERG1/β1 integrin cycle determines the slow time course and cyclic behavior of cell migration in cancer cells., (© 2023 Duranti et al.)

The human ether-à-go-go-related gene (hERG) is associated with drug cardiotoxicity. If the hERG channel is blocked, it will lead to prolonged QT interval and cause sudden death in severe cases. Therefore, it is important to evaluate the hERG-blocking property of compounds in early drug discovery. In this study, a dataset containing 4556 compounds with IC 50 values determined by patch clamp techniques on mammalian lineage cells was collected, and hERG blockers and non-blockers were distinguished according to three single thresholds and two binary thresholds. Four machine learning (ML) algorithms combining four molecular fingerprints and molecular descriptors as well as graph convolutional neural networks (GCNs) were used to construct a series of binary classification models. The results showed that the best models varied for different thresholds. The ML models implemented by support vector machine and random forest performed well based on Morgan fingerprints and molecular descriptors, with AUCs ranging from 0.884 to 0.950. GCN showed superior prediction performance with AUCs above 0.952, which might be related to its direct extraction of molecular features from the original input. Meanwhile, the classification of binary threshold was better than that of single threshold, which could provide us with a more accurate prediction of hERG blockers. At last, the applicability domain for the model was defined, and seven structural alerts that might generate hERG blockage were identified by information gain and substructure frequency analysis. Our work would be beneficial for identifying hERG blockers in chemicals., (© 2023 John Wiley & Sons Ltd.)

Introduction: Long QT Syndrome (LQTS) is an inherited disease with an abnormal electrical conduction system in the heart that can cause sudden death as a result of QT prolongation. LQT2 is the second most common subtype of LQTS caused by loss of function mutations in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. Although more than 900 mutations are associated with the LQTS, many of these mutations are not validated or characterized., Methods and Results: Sequencing analyses of genomic DNA of a family with LQT2 identified a putative mutation. i.e., KCNH2(NM_000238.3): c.3099_3112del, in KCNH2 gene which appeared to be a definite pathogenic mutation. The family pedigree information showed a gender difference in clinical features and T-wave morphology between male and female patients. The female with mutation exhibited recurring ventricular arrhythmia and syncope, while two male carriers did not show any symptoms. In addition, T-wave in females was much flatter than in males. The female proband showed a positive reaction to the lidocaine test. Lidocaine injection almost completely blocked ventricular arrhythmia and shortened the QT interval by ≥30 ms. Treatment with propranolol, mexiletine, and implantation of cardioverter-defibrillators prevented the sustained ventricular tachycardia, ventricular fibrillation, and syncope, as assessed by a 3-year follow-up evaluation., Conclusions: A putative mutation c.3099_3112del in the KCNH2 gene causes LQT2 syndrome, and the pathogenic mutation mainly causes symptoms in female progeny., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2023 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Abstract: Remdesivir, approved for the treatment of COVID-19, has been associated with heart-rate corrected QT interval (QTc) prolongation and torsade de pointes in case reports. However, data are conflicting regarding the ability of remdesivir to inhibit the human ether-a-go-go-related gene (hERG) -related current. The objective of this study was to investigate the effects remdesivir and its primary metabolite, GS-441524, on hERG-related currents. Human embryonic kidney 293 cells stably expressing hERG were treated with various concentrations of remdesivir and GS-441524. The effects of acute and prolonged exposure on hERG-related current were assessed using whole-cell configuration of voltage-clamp protocols. Acute exposure to remdesivir and GS-441524 had no effect on hERG currents and the half-activation voltage (V 1/2 ). Prolonged treatment with 100 nM and 1 µM remdesivir significantly reduced peak tail currents and hERG current density. The propensity for remdesivir to prolong QTc intervals and induce torsade de pointes in predisposed patients warrants further investigation., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Long QT syndrome type 2 (LQT2) is a genetic disorder caused by mutations in the KCNH2 gene, also known as the human ether-a-go-go-related gene (HERG). More than 30% of HERG mutations result in a premature termination codon that triggers a process called nonsense-mediated messenger RNA (mRNA) decay (NMD), where the mRNA transcript is degraded. NMD is a quality control mechanism that removes faulty mRNA to prevent the translation of truncated proteins. Recent advances in antisense oligonucleotide (ASO) technology in the field of cystic fibrosis (CF) have yielded significant progress, including the ASO-mediated comprehensive characterization of key NMD factors and exon-skipping therapy. These advances have contributed to our understanding of the role of premature termination codon-containing mutations in disease phenotypes and have also led to the development of potentially useful therapeutic strategies. Historically, studies of CF have provided valuable insights for the research on LQT2, particularly concerning increasing the expression of HERG. In this article, we outline the current state of knowledge regarding ASO, NMD, and HERG and discuss the introduction of ASO technology in the CF to elucidate the pathogenic mechanisms through targeting NMD. We also discuss the potential clinical therapeutic benefits and limitations of ASO for the management of LQT2. By drawing on lessons learned from CF research, we explore the potential translational values of these advances into LQT2 studies., (Copyright © 2023 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Potassium channels are often dysregulated in tumours of the gastrointestinal (GI) tract. Among them, the voltage-dependent channel K V 11.1, also known as human ether-à-go-go related gene 1 (hERG1), is frequently expressed in tumours and precancerous lesions of the GI tract. In precancerous lesions, hERG1 behaves as a progression factor, contributing to identifying those patients whose lesions can progress towards true cancers. In advanced cancers, such as colorectal and pancreatic cancer, a high hERG1 expression represents a negative prognostic factor, contributing to identifying high risk patients. The only exception is represented by neuroendocrine cancers of both the ileum and the pancreas, where hERG1 represents a positive prognostic factor for survival. In GI tumours, hERG1 can function either as a true channel, allowing outward potassium ion flux and membrane repolarisation, or in a non-canonical, non-conductive way. This occurs because, in cancer, hERG1 forms complexes with different plasma membrane and cytosolic proteins, instead of classical accessory subunits. In particular, hERG1 forms a complex with the β1 subunit of integrin receptors: the hERG1-β1 complex. Growth and chemokine receptors, small GTPases, phosphoinositide 3-kinase, as well as other ion transporters or channels, are also recruited in the hERG1-β1 complex. The formation of multiprotein channel complexes represents an emerging mechanism allowing functional channel networking in both excitable and non-excitable cells. hERG1 represents a prototype of how multiprotein complexes operate in tumours, that is, giving rise to signalling hubs which can transmit and modulate signals arising from the tumour microenvironment, hence contributing to tumour progression and malignancy., (© 2022 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Background: Long-QT syndrome (LQTS) is characterized by QT prolongation and increased risk for syncope, seizures, and sudden cardiac death. The majority of LQTS stems from pathogenic mutations in KCNQ1 , KCNH2 , or SCN5A . However, ≈10% of patients with LQTS remain genetically elusive. We utilized genome sequencing to identify a novel LQTS genetic substrate in a multigenerational genotype-negative LQTS pedigree., Methods: Genome sequencing was performed on 5 affected family members. Only rare nonsynonymous variants present in all affected family members were considered. The candidate variant was characterized functionally in patient-derived induced pluripotent stem cell and gene-edited, variant corrected, isogenic control induced pluripotent stem cell-derived cardiomyocytes., Results: A missense variant (p.G6S) was identified in ALG10B -encoded α-1,2-glucosyltransferase B protein. ALG10B (alpha-1,2-glucosyltransferase B protein) is a known interacting protein of KCNH2 -encoded K v 11.1 (HERG [human Ether-à-go-go-related gene]). Compared with isogenic control, ALG10B-p.G6S induced pluripotent stem cell-derived cardiomyocytes showed (1) decreased protein expression of ALG10B (p.G6S, 0.7±0.18, n=8 versus control, 1.25±0.16, n=9; P <0.05), (2) significant retention of HERG in the endoplasmic reticulum ( P <0.0005), and (3) a significantly prolonged action potential duration confirmed by both patch clamp (p.G6S, 531.1±38.3 ms, n=15 versus control, 324.1±21.8 ms, n=13; P <0.001) and multielectrode assay ( P <0.0001). Lumacaftor-a compound known to rescue HERG trafficking-shortened the pathologically prolonged action potential duration of ALG10B-p.G6S induced pluripotent stem cell-derived cardiomyocytes by 10.6% (n=31 electrodes; P <0.001)., Conclusions: Here, we demonstrate that ALG10B-p.G6S downregulates ALG10B, resulting in defective HERG trafficking and action potential duration prolongation. Therefore, ALG10B is a novel LQTS-susceptibility gene underlying the LQTS phenotype observed in a multigenerational pedigree. ALG10B mutation analysis may be warranted, especially in genotype-negative patients with an LQT2-like phenotype.

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

Background: Variants in KCNH2, encoding the human ether a-go-go (hERG) channel that is responsible for the rapid component of the cardiac delayed rectifier K+ current (IKr), are causal to long QT syndrome type 2 (LQTS2). We identified eight index patients with a new variant of unknown significance (VUS), KCNH2:c.2717C > T:p.(Ser906Leu). We aimed to elucidate the biophysiological effect of this variant, to enable reclassification and consequent clinical decision-making., Methods: A genotype-phenotype overview of the patients and relatives was created. The biophysiological effects were assessed independently by manual-, and automated calibrated patch clamp. HEK293a cells expressing (i) wild-type (WT) KCNH2, (ii) KCNH2-p.S906L alone (homozygous, Hm) or (iii) KCNH2-p.S906L in combination with WT (1:1) (heterozygous, Hz) were used for manual patching. Automated patch clamp measured the variants function against known benign and pathogenic variants, using Flp-In T-rex HEK293 KCNH2-variant cell lines., Results: Incomplete penetrance of LQTS2 in KCNH2:p.(Ser906Leu) carriers was observed. In addition, some patients were heterozygous for other VUSs in CACNA1C, PKP2, RYR2 or AKAP9. The phenotype of carriers of KCNH2:p.(Ser906Leu) ranged from asymptomatic to life-threatening arrhythmic events. Manual patch clamp showed a reduced current density by 69.8 and 60.4% in KCNH2-p.S906L-Hm and KCNH2-p.S906L-Hz, respectively. The time constant of activation was significantly increased with 80.1% in KCNH2-p.S906L-Hm compared with KCNH2-WT. Assessment of KCNH2-p.S906L-Hz by calibrated automatic patch clamp assay showed a reduction in current density by 35.6%., Conclusion: The reduced current density in the KCNH2-p.S906L-Hz indicates a moderate loss-of-function. Combined with the reduced penetrance and variable phenotype, we conclude that KCNH2:p.(Ser906Leu) is a low penetrant likely pathogenic variant for LQTS2., (© The Author(s) 2022. Published by Oxford University Press.)

KCNH2 encodes the human ether-a-go-go-related gene (hERG) potassium channel and is an important repolarization reserve for regulating cardiac electrical activity. Increasing evidence suggests that it is involved in the development of various tumours, yet a thorough analysis of the underlying process has not been performed. Here, we have comprehensively examined the role of KCNH2 in multiple cancers by assessing KCNH2 gene expression, diagnostic and prognostic value, genetic alterations, immune infiltration correlations, RNA modifications, mutations, clinical correlations, interacting proteins, and associated signalling pathways. KCNH2 is differentially expressed in over 30 cancers and has a high diagnostic value for 10 tumours. Survival analysis showed that high expression of KCNH2 was associated with a poor prognosis in glioblastoma multiforme (GBM) and hepatocellular carcinoma (LIHC). Mutations and RNA methylation modifications (especially m6A) of KCNH2 are associated with its expression in multiple tumours. KCNH2 expression is correlated with tumour mutation burden, microsatellite instability, neoantigen load, and mutant-allele tumour heterogeneity. In addition, KCNH2 expression is associated with the tumour immune microenvironment and its immunosuppressive phenotype. KEGG signalling pathway enrichment analysis revealed that KCNH2 and its interacting molecules are involved in a variety of pathways related to carcinogenesis and signal regulation, such as the PI3K/Akt and focal adhesion pathways. Overall, we found that KCNH2 and its interaction molecular are expected to be immune-related biomarkers for cancer diagnosis and prognosis evaluation, and are potential regulatory targets of singalling pathways for tumour development due to their significant role in cancers., (© 2023. The Author(s).)

Background and Objectives: KCNH5 encodes the voltage-gated potassium channel EAG2/Kv10.2. We aimed to delineate the neurodevelopmental and epilepsy phenotypic spectrum associated with de novo KCNH5 variants., Methods: We screened 893 individuals with developmental and epileptic encephalopathies for KCNH5 variants using targeted or exome sequencing. Additional individuals with KCNH5 variants were identified through an international collaboration. Clinical history, EEG, and imaging data were analyzed; seizure types and epilepsy syndromes were classified. We included 3 previously published individuals including additional phenotypic details., Results: We report a cohort of 17 patients, including 9 with a recurrent de novo missense variant p.Arg327His, 4 with a recurrent missense variant p.Arg333His, and 4 additional novel missense variants. All variants were located in or near the functionally critical voltage-sensing or pore domains, absent in the general population, and classified as pathogenic or likely pathogenic using the American College of Medical Genetics and Genomics criteria. All individuals presented with epilepsy with a median seizure onset at 6 months. They had a wide range of seizure types, including focal and generalized seizures. Cognitive outcomes ranged from normal intellect to profound impairment. Individuals with the recurrent p.Arg333His variant had a self-limited drug-responsive focal or generalized epilepsy and normal intellect, whereas the recurrent p.Arg327His variant was associated with infantile-onset DEE. Two individuals with variants in the pore domain were more severely affected, with a neonatal-onset movement disorder, early-infantile DEE, profound disability, and childhood death., Discussion: We describe a cohort of 17 individuals with pathogenic or likely pathogenic missense variants in the voltage-sensing and pore domains of Kv10.2, including 14 previously unreported individuals. We present evidence for a putative emerging genotype-phenotype correlation with a spectrum of epilepsy and cognitive outcomes. Overall, we expand the role of EAG proteins in human disease and establish KCNH5 as implicated in a spectrum of neurodevelopmental disorders and epilepsy., (© 2022 American Academy of Neurology.)

Cervical cancer is the fourth most common cancer among women worldwide. The main factor associated with the onset and progression of this neoplasia is the human papillomavirus (HPV) infection. The HPV-oncogenes E6 and E7 are critical drivers of cellular transformation, promoting the expression of oncogenes such as KCNH1 . The phytochemical α-mangostin (AM) is a potent antineoplastic and antiviral compound. However, its effects on HPV oncogenes and KCNH1 gene expression remain unknown. This study evaluated the effects of AM on cell proliferation, cell cycle distribution and gene expression, including its effects on tumor growth in xenografted mice. AM inhibited cell proliferation in a concentration-dependent manner, being the most sensitive cell lines those with the highest number of HPV16 copies. In addition, AM promoted G1-cell cycle arrest in CaSki cells, while led to cell death in SiHa and HeLa cells. Of interest was the finding of an AM-dependent decreased gene expression of E6, E7 and KCNH1 both in vitro and in vivo, as well as the modulation of cytokine expression, Ki-67, and tumor growth inhibition. On these bases, we suggest that AM represents a good option as an adjuvant for the treatment and prevention of cervical cancer.

Gemcitabine is an antineoplastic drug commonly used in the treatment of several types of cancers including pancreatic cancer and non-small cell lung cancer. Although gemcitabine-induced cardiotoxicity is widely recognized, the exact mechanism of cardiac dysfunction causing arrhythmias remains unclear. The objective of this study was to electrophysiologically evaluate the proarrhythmic cardiotoxicity of gemcitabine focusing on the human rapid delayed rectifier potassium channel, hERG channel. In heterologous hERG expressing HEK293 cells (hERG-HEK cells), hERG channel current (IhERG) was reduced by gemcitabine when applied for 24 h but not immediately after the application. Gemcitabine modified the activation gating properties of the hERG channel toward the hyperpolarization direction, while inactivation, deactivation or reactivation gating properties were unaffected by gemcitabine. When gemcitabine was applied to hERG-HEK cells in combined with tunicamycin, an inhibitor of N-acetylglucosamine phosphotransferase, gemcitabine was unable to reduce IhERG or shift the activation properties toward the hyperpolarization direction. While a mannosidase I inhibitor kifunensine alone reduced IhERG and the reduction was even larger in combined with gemcitabine, kifunensine was without effect on IhERG when hERG-HEK cells were pretreated with gemcitabine for 24 h. In addition, gemcitabine down-regulated fluorescence intensity for hERG potassium channel protein in rat neonatal cardiomyocyte, although hERG mRNA was unchanged. Our results suggest the possible mechanism of arrhythmias caused by gemcitabine revealing a down-regulation of IhERG through the post-translational glycosylation disruption possibly at the early phase of hERG channel glycosylation in the endoplasmic reticulum that alters the electrical excitability of cells., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Wei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In addition, to their established role in cardiac myocytes and neurons, ion channels encoded by ether-a-go-go-related genes (ERG1-3 or kcnh2,3 and 6) (kcnh2) are functionally relevant in phasic smooth muscle. The aim of the study was to determine the expression and functional impact of ERG expression products in rat urinary bladder smooth muscle using quantitative polymerase chain reaction, immunocytochemistry, whole-cell patch-clamp and isometric tension recording. kcnh2 was expressed in rat bladder, whereas kcnh6 and kcnh3 expression were negligible. Immunofluorescence for the kcnh2 expression product Kv11.1 was detected in the membrane of isolated smooth muscle cells. Potassium currents with voltage-dependent characteristics consistent with Kv11.1 channels and sensitive to the specific blocker E4031 (1 μM) were recorded from isolated detrusor smooth muscles. Disabling Kv11.1 activity with specific blockers (E4031 and dofetilide, 0.2-20 μM) augmented spontaneous contractions to a greater extent than BK Ca channel blockers, enhanced carbachol-driven activity, increased nerve stimulation-mediated contractions, and impaired β-adrenoceptor-mediated inhibitory responses. These data establish for the first time that Kv11.1 channels are key determinants of contractility in rat detrusor smooth muscle., (© 2023 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Most blockers of both hERG (human ether-à-go-go-related gene) channels and pancreatic ß-cell ATP-sensitive K + (K ATP ) channels access their binding sites from the cytoplasmic side of the plasma membrane. It is unknown whether binding to intracellular components competes with binding of these substances to K + channels. The whole-cell configuration of the patch-clamp technique, a laser-scanning confocal microscope, and fluorescence correlation spectroscopy (FCS) were used to study hERG channels expressed in HEK (human embryonic kidney) 293 cells and K ATP channels from the clonal insulinoma cell line RINm5F. When applied via the pipette solution in the whole-cell configuration, terfenadine blocked both hERG and K ATP currents with much lower potency than after application via the bath solution, which was not due to P-glycoprotein-mediated efflux of terfenadine. Such a difference was not observed with dofetilide and tolbutamide. 37-68% of hERG/EGFP (enhanced green-fluorescent protein) fusion proteins expressed in HEK 293 cells were slowly diffusible as determined by laser-scanning microscopy in the whole-cell configuration and by FCS in intact cells. Bath application of a green-fluorescent sulphonylurea derivative (Bodipy-glibenclamide) induced a diffuse fluorescence in the cytosol of RINm5F cells under whole-cell patch-clamp conditions. These observations demonstrate the presence of intracellular binding sites for hERG and K ATP channel blockers not dialyzable by the patch-pipette solution. Intracellular binding of terfenadine was not influenced by a mutated hERG (Y652A) channel. In conclusion, substances with high lipophilicity are not freely diffusible inside the cell but steep concentration gradients might exist within the cell and in the sub-membrane space., (© 2022. The Author(s).)

KCNH2 encodes hERG1, the voltage-gated potassium channel that conducts the rapid delayed rectifier potassium current (I Kr ) in human cardiac tissue. hERG1 is one of the first channels expressed during early cardiac development, and its dysfunction is associated with intrauterine fetal death, sudden infant death syndrome, cardiac arrhythmia, and sudden cardiac death. Here, we identified a hERG1 polypeptide (hERG1 NP ) that is targeted to the nuclei of immature cardiac cells, including human stem cell-derived cardiomyocytes (hiPSC-CMs) and neonatal rat cardiomyocytes. The nuclear hERG1 NP immunofluorescent signal is diminished in matured hiPSC-CMs and absent from adult rat cardiomyocytes. Antibodies targeting distinct hERG1 channel epitopes demonstrated that the hERG1 NP signal maps to the hERG1 distal C-terminal domain. KCNH2 deletion using CRISPR simultaneously abolished I Kr and the hERG1 NP signal in hiPSC-CMs. We then identified a putative nuclear localization sequence (NLS) within the distal hERG1 C-terminus, 883-RQRKRKLSFR-892. Interestingly, the distal C-terminal domain was targeted almost exclusively to the nuclei when overexpressed HEK293 cells. Conversely, deleting the NLS from the distal peptide abolished nuclear targeting. Similarly, blocking α or β1 karyopherin activity diminished nuclear targeting. Finally, overexpressing the putative hERG1 NP peptide in the nuclei of HEK cells significantly reduced hERG1a current density, compared to cells expressing the NLS-deficient hERG1 NP or GFP. These data identify a developmentally regulated polypeptide encoded by KCNH2 , hERG1 NP , whose presence in the nucleus indirectly modulates hERG1 current magnitude and kinetics.

The human ether-a-go-go-related gene (hERG) K + channel conducts a rapidly activating delayed rectifier K + current (I Kr ), which is essential for normal electrical activity of the heart. Precise regulation of hERG channel biogenesis is critical for serving its physiological functions, and deviations from the regulation result in human diseases. However, the mechanism underlying the precise regulation of hERG channel biogenesis remains elusive. Here, by using forward genetic screen, we found that PATR-1, the Caenorhabditis elegans homolog of the yeast DNA topoisomerase 2-associated protein PAT1, is a critical regulator for the biogenesis of UNC-103, the ERG K + channel in C. elegans . A loss-of-function mutation in patr-1 down-regulates the expression level of UNC-103 proteins and suppresses the phenotypic defects resulted from a gain-of-function mutation in the unc-103 gene. Furthermore, downregulation of PATL1 and PATL2, the human homologs of PAT1, decreases protein levels and the current density of native hERG channels in SH-SY5Y cells and human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Knockdown of PATL1 and PATL2 elongates the duration of action potentials in hiPSC-CMs, suggesting that PATL1 and PATL2 affect the function of hERG channels and hence electrophysiological characteristics in the human heart. Further studies found that PATL1 and PATL2 interact with TFIIE, a general transcription factor required for forming the RNA polymerase II preinitiation complex, and dual-luciferase reporter assays indicated that PATL1 and PATL2 facilitate the transcription of hERG mRNAs. Together, our study discovers that evolutionarily conserved DNA topoisomerase 2-associated proteins regulate the biogenesis of hERG channels via a transcriptional mechanism.

Fast C-type inactivation confers distinctive functional properties to the hERG potassium channel, and its association to inherited and acquired cardiac arrythmias makes the study of the inactivation mechanism of hERG at the atomic detail of paramount importance. At present, two models have been proposed to describe C-type inactivation in K + -channels. Experimental data and computational work on the bacterial KcsA channel support the hypothesis that C-type inactivation results from a closure of the selectivity filter that sterically impedes ion conduction. Alternatively, recent experimental structures of a mutated Shaker channel revealed a widening of the extracellular portion of the selectivity filter, which might diminish conductance by interfering with the mechanism of ion permeation. Here, we performed molecular dynamics simulations of the wild-type hERG, a non-inactivating mutant (hERG-N629D), and a mutant that inactivates faster than the wild-type channel (hERG-F627Y) to find out which and if any of the two reported C-type inactivation mechanisms applies to hERG. Closure events of the selectivity filter were not observed in any of the simulated trajectories but instead, the extracellular section of the selectivity filter deviated from the canonical conductive structure of potassium channels. The degree of widening of the potassium binding sites at the extracellular entrance of the channel was directly related to the degree of inactivation with hERG-F627Y > wild-type hERG > hERG-N629D. These findings support the hypothesis that C-type inactivation in hERG entails a widening of the extracellular entrance of the channel rather than a closure of the selectivity filter.

Purpose: Genotype-phenotypic correlation of KCNH1 variant remains elusive. This study aimed to expand the phenotypic spectrum of KCNH1 and explore the correlations between epilepsy and molecular sub-regional locations., Methods: We performed whole-exome sequencing in a cohort of 98 patients with familiar febrile seizure (FS) or epilepsy with unexplained etiologies. The damaging effects of variants were predicted by protein modeling and multiple in silico tools. All reported patients with KCNH1 pathogenic variants with detailed neurological phenotypes were analyzed to evaluate the genotype-phenotype correlation., Results: Two novel KCNH1 variants were identified in three cases, including two patients with FS with inherited variant (p.Ile113Thr) and one boy with epilepsy with de novo variant (p.Arg357Trp). Variant Ile113Thr was located within the eag domain, and variant p.Arg357Trp was located in transmembrane domain 4 of KCNH1, respectively. Two patients experienced refractory status epilepticus (SE), of which one patient died of acute encephalopathy induced by SE. Further analysis of 30 variants in 51 patients demonstrated that de novo variants were associated with epileptic encephalopathy, while mosaic/somatic or germline variants cause isolated epilepsy/FS. All hotspot variants associated with epileptic encephalopathy clustered in transmembrane domain (S4 and S6), while those with isolated epilepsy/seizures or TBS/ZLS without epilepsy were scattered in the KCNH1., Conclusions: We found two novel missense variants of KCNH1 in three individuals with isolated FS/epilepsy. Variants in the KCNH1 cause a spectrum of epileptic disorders ranging from a benign form of genetic isolated epilepsy/FS to intractable form of epileptic encephalopathy. The genotypes and variant locations help explaining the phenotypic variation of patients with KCNH1 variant., (© 2022 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Introduction: The coronavirus disease 2019 (COVID-19) pandemic has led to millions of confirmed cases and deaths worldwide and has no approved therapy. Currently, more than 700 drugs are tested in the COVID-19 clinical trials, and full evaluation of their cardiotoxicity risks is in high demand., Methods: We mainly focused on hydroxychloroquine (HCQ), one of the most concerned drugs for COVID-19 therapy, and investigated the effects and underlying mechanisms of HCQ on hERG channel via molecular docking simulations. We further applied the HEK293 cell line stably expressing hERG-wild-type channel (hERG-HEK) and HEK293 cells transiently expressing hERG-p.Y652A or hERG-p.F656A mutants to validate our predictions. Western blot analysis was used to determine the hERG channel, and the whole-cell patch clamp was utilized to record hERG current (IhERG)., Results: HCQ reduced the mature hERG protein in a time- and concentration-dependent manner. Correspondingly, chronic and acute treatment of HCQ decreased the hERG current. Treatment with brefeldin A (BFA) and HCQ combination reduced hERG protein to a greater extent than BFA alone. Moreover, disruption of the typical hERG binding site (hERG-p.Y652A or hERG-p.F656A) rescued HCQ-mediated hERG protein and IhERG reduction., Conclusion: HCQ can reduce the mature hERG channel expression and IhERG via enhancing channel degradation. The QT prolongation effect of HCQ is mediated by typical hERG binding sites involving residues Tyr652 and Phe656., (© 2023 S. Karger AG, Basel.)

Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include 'difficult-to-express' membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) K V 11.1 and the voltage-gated sodium channel hNa V 1.5., (© 2023. The Author(s).)

Due to challenges with historical data and the diversity of assay formats, in silico models for safety-related endpoints are often based on discretized data instead of the data on a natural continuous scale. Models for discretized endpoints have limitations in usage and interpretation that can impact compound design. Here, we present a consistent data inference approach, exemplified on two data sets of Ether-à-go-go-Related Gene (hERG) K+ inhibition data, for dose-response and screening experiments that are generally applicable for in vitro assays. hERG inhibition has been associated with severe cardiac effects and is one of the more prominent safety targets assessed in drug development, using a wide array of in vitro and in silico screening methods. In this study, the IC 50 for hERG inhibition is estimated from diverse historical proprietary data. The IC 50 derived from a two-point proprietary screening data set demonstrated high correlation (R = 0.98, MAE = 0.08) with IC 50s derived from six-point dose-response curves. Similar IC 50 estimation accuracy was obtained on a public thallium flux assay data set (R = 0.90, MAE = 0.2). The IC 50 data were used to develop a robust quantitative model. The model's MAE (0.47) and R 2 (0.46) were on par with literature statistics and approached assay reproducibility. Using a continuous model has high value for pharmaceutical projects, as it enables rank ordering of compounds and evaluation of compounds against project-specific inhibition thresholds. This data inference approach can be widely applicable to assays with quantitative readouts and has the potential to impact experimental design and improve model performance, interpretation, and acceptance across many standard safety endpoints.

Locus coeruleus (LC) neurons, with their extensive innervations throughout the brain, control a broad range of physiological processes. Several ion channels have been characterized in LC neurons that control intrinsic membrane properties and excitability. However, ERG ( ether-à-go-go-related gene ) K + channels that are particularly important in setting neuronal firing rhythms and automaticity have not as yet been discovered in the LC. Moreover, the neurophysiological and pathophysiological roles of ERG channels in the brain remain unclear despite their expression in several structures. By performing immunohistochemical investigations, we found that ERG-1A, ERG-1B, ERG-2 and ERG-3 are highly expressed in the LC neurons of mice. To examine the functional role of ERG channels, current-clamp recordings were performed on mouse LC neurons in brain slices under visual control. ERG channel blockade by WAY-123,398, a class III anti-arrhythmic agent, increased the spontaneous firing activity and discharge irregularity of LC neurons. Here, we have shown the presence of distinct ERG channel subunits in the LC which play an imperative role in modulating neuronal discharge patterns. Thus, we propose that ERG channels are important players behind the changes in, and/or maintenance of, LC firing patterns that are implicated in the generation of different behaviors and in several disorders.

Abstract: Tolterodine is a first-line antimuscarinic drug used to treat overactive bladder. Adverse cardiac effects including tachycardia and palpitations have been observed, presumably because of its inhibition of the human ether-à-go-go-related gene (hERG) K + channel. However, the molecular mechanism of hERG channel inhibition by tolterodine is largely unclear. In this study, we performed molecular docking to identify potential binding sites of tolterodine in hERG channel, and two-microelectrode voltage-clamp to record the currents of hERG and its mutants expressed in Xenopus oocytes. The results of computational modeling demonstrated that phenylalanine at position 656 (F656) and tyrosine at position 652 (Y652) on the S6 helix of hERG channel are the most favorable binding residues of tolterodine, which was validated by electrophysiological recordings on Y652A and F656A hERG mutants. The Y652A and F656A mutations decreased inhibitory potency of tolterodine 345-fold and 126-fold, respectively. The Y652A mutation significantly altered the voltage dependence of channel inhibition by tolterodine. For both the wild-type and the mutant channels, tolterodine reduced the currents in a time-dependent manner, and the blockade occurred with the channel activated. Tolterodine did not interfere with hERG channel deactivation, whereas channel inactivation greatly impaired its blocking effect. The inhibition of hERG channel by tolterodine is independent of its action on muscarinic acetylcholine receptors. In conclusion, tolterodine is an open-state blocker of hERG K + channel with nanomolar potency. Y652 and F656, 2 aromatic residues on the inner S6 helix, are responsible for the high-affinity binding of tolterodine to hERG channel., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Clinically, hypoxia is a major risk factor for long QT syndrome (LQTS), which is associated with many diseases, such as myocardial ischemia. LQTS can be caused by the deficiency of hERG, a potassium ion channel that plays a key role in cardiac repolarization. Modifications such as acetylation of histones or non-histone proteins can affect the protein expression. In the present study, we explored the mechanism underlying hypoxia-induced LQTS and a potential reversal strategy. Experiments were performed under hypoxia to determine transcriptional and post-transcriptional expression changes. We used real-time PCR, chromatin immunoprecipitation assay, and western blotting to determine the histones acetylation in the hERG gene and the mechanism. Molecular docking, western blotting, IP, and patch -clamp assay were performed to determine the acetylation and ubiquitination levels of hERG protein and the mechanism. hERG mRNA and protein expression were found to decrease under hypoxia. The histone deacetylation level increased under hypoxia at both H3K27 and H4 of the hERG gene. HDAC1 and HDAC2 are the key enzymes for the mechanism. HDAC6 directly interacts with hERG. The acetylation level of hERG decreased and the ubiquitination level of hERG increased under hypoxia. The inhibitors of HDAC1, HDAC2, and HDAC6 could reverse the reduction of hERG mRNA and hERG protein expression under hypoxia. In conclusion, deacetylation of hERG gene-associated histones and hERG protein might be the mechanisms for LQTS in patients with hypoxia, and the inhibition of HDAC1, HDAC2, and HDAC6 might be a promising reversal strategy for reducing hERG expression under different pathological conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Mutations in KCNH6 has been proved to cause hypoinsulinemia and diabetes in human and mice. Cisapride is a stomach-intestinal motility drug used to treat gastrointestinal dysfunction. Cisapride has been reported to be a potential inhibitor of the KCNH family, but it remained unclear whether cisapride inhibited KCNH6. Here, we discovered the role of cisapride on glucose metabolism, focusing on the KCNH6 potassium channel protein. Cisapride reduced blood glucose level and increased serum insulin secretion in wild-type (WT) mice fed standard normal chow/a high-fat diet or in db/db mice, especially when combined with tolbutamide. This effect was much stronger after 4 weeks of intraperitoneal injection. Whole-cell patch-clamp showed that cisapride inhibited KCNH6 currents in transfected HEK293 cells in a concentration-dependent manner. Cisapride induced an increased insulin secretion through the disruption of intracellular calcium homeostasis in a rat pancreatic β-cell line, INS-1E. Further experiments revealed that cisapride did not decrease blood glucose or increase serum insulin in KCNH6 β-cell knockout (Kcnh6-β-KO) mice when compared with WT mice. Cisapride also ameliorated glucose-stimulated insulin secretion (GSIS) in response to high glucose in WT but not Kcnh6-β-KO mice. Thus, our data reveal a novel way for the effect of KCNH6 in cisapride-induced hypoglycemia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lu, Shi, Yuan, Xie, Zhao, Zhu and Yang.)

hERG1 potassium channels are widely expressed in human cancers of different origins, where they affect several key aspects of cellular behaviour. The present study was designed to evaluate the expression and clinical relevance of hERG1 protein in cancer tissues from patients suffering from neuroendocrine tumours (NETs) of ileal (iNETs) and pancreatic (pNETs) origin, with available clinicopathological history and follow-up. The study was carried out by immunohistochemistry with an anti-hERG1 monoclonal antibody. In a subset of samples, a different antibody directed against the hERG1/β1 integrin complex was also used. The analysis showed for the first time that hERG1 is expressed in human NETs originating from either the ileum or the pancreas. hERG1 turned out to have a prognostic value in NETs, showing (i) a statistically significant positive impact on OS of patients affected by ileal NETs, regardless the TNM stage; (ii) a statistically significant positive impact on OS of patients affected by aggressive (TNM stage IV) disease, either ileal or pancreatic; (iii) a trend to a negative impact on OS of patients affected by less aggressive (TNM stage I-III) disease, either ileal or pancreatic. Moreover, in order to evaluate whether ERG1 was functionally expressed in a cellular model of pNET, the INS1E rat insulinoma cell line was used, and it emerged that blocking ERG1 with a specific inhibitor of the channel (E4031) turned out in a significant reduction in cell proliferation.

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 K r 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: Gingerols are bioactive compounds derived from ginger, our experiment investigates the effects of 6-, 8- and 10-Gin on the human ether-à-go-go-related gene (hERG) K+ channels by using patch clamp technology., Key Findings: hERG K+ currents were suppressed by 6-, 8- and 10-Gin in a concentration-dependent manner. The IC50 values of 6-, 8- and 10-Gin were 41.5, 16.1 and 86.5 μM for the hERG K+ currents, respectively. The maximum inhibitory effects caused by 6-, 8- and 10-Gin were 44.3% ± 2.0%, 88.6% ± 1.3% and 63.1% ± 1.1%, respectively, and the effects were almost completely reversible., Conclusion: These findings suggest that 8-Gin is the most potent hERG K+ channel inhibitor among gingerol components and may offer a new approach for understanding and treating cancer., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Evaluation of cardiotoxicity potential of new chemical entities (NCEs) has lately become one of the stringent filters in the drug discovery and development process. Cardiotoxicity is caused mainly by the inhibition of human ether-a-go-go related gene (hERG) channel protein. Inhibition of the hERG channel leads to a life-threatening condition known as cardiac arrhythmia. Knowledge of the structural behaviour of the hERG would aid greatly in the design of new drug molecules that do not interact with the protein and add to the safety index. In this study, a computational model for the active-state of hERG was developed. This model was equilibrated by performing the molecular dynamics simulations for 100 ns followed by clustering and selection of a representative structure based on the largest populated cluster. To study the changes in the protein structure on inhibition, three inhibitory ligands, namely, dofetilide, cisapride and terfenadine were docked, followed by molecular dynamics simulations of 200 ns for the apo and each ligand-bound structure. It was observed that docking and simulation studies of the hERG model exhibited noticeable conformational changes in the protein upon ligand-binding. A significant change in the kink of the S6-transmembrane helix was observed. Inter-chain distances between the crucial residues Y652 and F656 (present below the ion-selectivity filter), their side-chain orientation and hydrogen bonding indicated a probable collapse of the pore. These changes may infer the initiation in transition of hERG from an open to an inactive state. Hence, these findings would help in designing compounds devoid of hERG inhibition with reduced cardiotoxicity.Communicated by Ramaswamy H. Sarma.

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP&#95;002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging., (© 2022. The Author(s).)

Carcinogenesis is a multistage process involving the dysregulation of multiple genes, proteins, and pathways that make any normal cell acquire a cancer cell phenotype. Therefore, it is no surprise that numerous ion channels could be involved in this process. Since their discovery and subsequent cloning, ion channels have been established as therapeutic targets in excitable cell pathologies (e.g., cardiac arrhythmias or epilepsy); however, their involvement in non-excitable cell pathologies is relatively recent. Among all ion channels, the voltage-gated potassium channels Kv10.1 have been established as a promising target in cancer treatment due to their high expression in tumoral tissues compared to low levels in healthy tissues.