Revealing a hidden conducting state by manipulating the intracellular domains in KV10.1 exposes the coupling between two gating mechanisms.

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(KV10.1) 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 Ca2+-Calmodulin and generate a model that describes the characteristic features of KCNH channels gating.

Potassium dependent structural changes in the selectivity filter of HERG potassium channels.

The fine tuning of biological electrical signaling is mediated by variations in the rates of opening and closing of gates that control ion flux through different ion channels. Human ether-a-go-go related gene (HERG) potassium channels have uniquely rapid inactivation kinetics which are critical to the role they play in regulating cardiac electrical activity. Here, we exploit the K+ sensitivity of HERG inactivation to determine structures of both a conductive and non-conductive selectivity filter structure of HERG. The conductive state has a canonical cylindrical shaped selectivity filter. The non-conductive state is characterized by flipping of the selectivity filter valine backbone carbonyls to point away from the central axis. The side chain of S620 on the pore helix plays a central role in this process, by coordinating distinct sets of interactions in the conductive, non-conductive, and transition states. Our model represents a distinct mechanism by which ion channels fine tune their activity and could explain the uniquely rapid inactivation kinetics of HERG.

Photoinhibition of the hERG potassium channel PAS domain by ultraviolet light speeds channel closing.

hERG potassium channels are critical for cardiac excitability. hERG channels have a Per-Arnt-Sim (PAS) domain at their N-terminus, and here, we examined the mechanism for PAS domain regulation of channel opening and closing (gating). We used TAG codon suppression to incorporate the noncanonical amino acid 4-benzoyl-L-phenylalanine (BZF), which is capable of forming covalent cross-links after photoactivation by ultraviolet (UV) light, at three locations (G47, F48, and E50) in the PAS domain. We found that hERG-G47BZF channels had faster closing (deactivation) when irradiated in the open state (at 0 mV) but showed no measurable changes when irradiated in the closed state (at -100 mV). hERG-F48BZF channels had slower activation, faster deactivation, and a marked rightward shift in the voltage dependence of activation when irradiated in the open (at 0 mV) or closed (at -100 mV) state. hERG-E50BZF channels had no measurable changes when irradiated in the open state (at 0 mV) but had slower activation, faster deactivation, and a rightward shift in the voltage dependence of activation when irradiated in the closed state (at -100mV), indicating that hERG-E50BZF had a state-dependent difference in UV photoactivation, which we interpret to mean that PAS underwent molecular motions between the open and closed states. Moreover, we propose that UV-dependent biophysical changes in hERG-G47BZF, F48BZF, and E50BZF were the direct result of photochemical cross-linking that reduced dynamic motions in the PAS domain and broadly stabilized the closed state relative to the open state of the channel.

The scorpion toxin BeKm-1 blocks hERG cardiac potassium channels using an indispensable arginine residue.

BeKm-1 is a peptide toxin from scorpion venom that blocks the pore of the potassium channel hERG (Kv11.1) in the human heart. Although individual protein structures have been resolved, the structure of the complex between hERG and BeKm-1 is unknown. Here, we used molecular dynamics and ensemble docking, guided by previous double-mutant cycle analysis data, to obtain an in silico model of the hERG-BeKm-1 complex. Adding to the previous mutagenesis study of BeKm-1, our model uncovers the key role of residue Arg20, which forms three interactions (a salt bridge and hydrogen bonds) with the channel vestibule simultaneously. Replacement of this residue even by lysine weakens the interactions significantly. In accordance, the recombinantly produced BeKm-1R20K mutant exhibited dramatically decreased activity on hERG. Our model may be useful for future drug design attempts.

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation.

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.

hERG1 channel subunit composition mediates proton inhibition of rapid delayed rectifier potassium current (IKr) in cardiomyocytes derived from hiPSCs.

The voltage-gated channel, hERG1, conducts the rapid delayed rectifier potassium current (IKr) and is critical for human cardiac repolarization. Reduced IKr causes long QT syndrome and increases the risk for cardiac arrhythmia and sudden death. At least two subunits form functional hERG1 channels, hERG1a and hERG1b. Changes in hERG1a/1b abundance modulate IKr kinetics, magnitude, and drug sensitivity. Studies from native cardiac tissue suggest that hERG1 subunit abundance is dynamically regulated, but the impact of altered subunit abundance on IKr and its response to external stressors is not well understood. Here, we used a substrate-driven human-induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) maturation model to investigate how changes in relative hERG1a/1b subunit abundance impact the response of native IKr to extracellular acidosis, a known component of ischemic heart disease and sudden infant death syndrome. IKr recorded from immatured hiPSC-CMs displays a 2-fold greater inhibition by extracellular acidosis (pH 6.3) compared with matured hiPSC-CMs. Quantitative RT-PCR and immunocytochemistry demonstrated that hERG1a subunit mRNA and protein were upregulated and hERG1b subunit mRNA and protein were downregulated in matured hiPSC-CMs compared with immatured hiPSC-CMs. The shift in subunit abundance in matured hiPSC-CMs was accompanied by increased IKr. Silencing hERG1b's impact on native IKr kinetics by overexpressing a polypeptide identical to the hERG1a N-terminal Per-Arnt-Sim domain reduced the magnitude of IKr proton inhibition in immatured hiPSC-CMs to levels comparable to those observed in matured hiPSC-CMs. These data demonstrate that hERG1 subunit abundance is dynamically regulated and determines IKr proton sensitivity in hiPSC-CMs.

Molecular dynamics simulations suggest possible activation and deactivation pathways in the hERG channel.

The elusive activation/deactivation mechanism of hERG is investigated, a voltage-gated potassium channel involved in severe inherited and drug-induced cardiac channelopathies, including the Long QT Syndrome. Firstly, the available structural data are integrated by providing a homology model for the closed state of the channel. Secondly, molecular dynamics combined with a network analysis revealed two distinct pathways coupling the voltage sensor domain with the pore domain. Interestingly, some LQTS-related mutations known to impair the activation/deactivation mechanism are distributed along the identified pathways, which thus suggests a microscopic interpretation of their role. Split channels simulations clarify a surprising feature of this channel, which is still able to gate when a cut is introduced between the voltage sensor domain and the neighboring helix S5. In summary, the presented results suggest possible activation/deactivation mechanisms of non-domain-swapped potassium channels that may aid in biomedical applications.

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

H1153Y-KCNH2 Mutation Identified in a Sudden Arrhythmic Death Syndrome Case Alters Channel Gating.

Long QT syndrome is one of the most common hereditary channelopathies inducing fatal arrhythmias and sudden cardiac death. We identified in a sudden arrhythmic death syndrome case a C-term KCNH2 mutation (c.3457C > T; p.His1153Tyr) classified as variant of unknown significance and functional impact. Heterologous expression in HEK293 cells combined with western-blot, flow-cytometry, immunocytochemical and microscope analyses shows no modification of channel trafficking to the cell membrane. Electrophysiological studies reveal that the mutation causes a loss of HERG channel function through an alteration of channel biophysical properties that reduces the current density leading to LQT2. These results provide the first functional evidence for H1153Y-KCNH2 mutation-induced abnormal channel properties. They concur with previous biophysical and clinical presentations of a survived patient with another variant that is G1036D. Therefore, the present report importantly highlights the potential severity of variants that may have useful implications for treatment, surveillance, and follow-up of LQT2 patients.

Machine learning models for classification tasks related to drug safety.

In this review, we outline the current trends in the field of machine learning-driven classification studies related to ADME (absorption, distribution, metabolism and excretion) and toxicity endpoints from the past six years (2015-2021). The study focuses only on classification models with large datasets (i.e. more than a thousand compounds). A comprehensive literature search and meta-analysis was carried out for nine different targets: hERG-mediated cardiotoxicity, blood-brain barrier penetration, permeability glycoprotein (P-gp) substrate/inhibitor, cytochrome P450 enzyme family, acute oral toxicity, mutagenicity, carcinogenicity, respiratory toxicity and irritation/corrosion. The comparison of the best classification models was targeted to reveal the differences between machine learning algorithms and modeling types, endpoint-specific performances, dataset sizes and the different validation protocols. Based on the evaluation of the data, we can say that tree-based algorithms are (still) dominating the field, with consensus modeling being an increasing trend in drug safety predictions. Although one can already find classification models with great performances to hERG-mediated cardiotoxicity and the isoenzymes of the cytochrome P450 enzyme family, these targets are still central to ADMET-related research efforts.

Flavonoids and hERG channels: Friends or foes?

The cardiac action potential is regulated by several ion channels. Drugs capable to block these channels, in particular the human ether-à-go-go-related gene (hERG) channel, also known as KV11.1 channel, may lead to a potentially lethal ventricular tachyarrhythmia called "Torsades de Pointes". Thus, evaluation of the hERG channel off-target activity of novel chemical entities is nowadays required to safeguard patients as well as to avoid attrition in drug development. Flavonoids, a large class of natural compounds abundantly present in food, beverages, herbal medicines, and dietary food supplements, generally escape this assessment, though consumed in consistent amounts. Continuously growing evidence indicates that these compounds may interact with the hERG channel and block it. The present review, by examining numerous studies, summarizes the state-of-the-art in this field, describing the most significant examples of direct and indirect inhibition of the hERG channel current operated by flavonoids. A description of the molecular interactions between a few of these natural molecules and the Rattus norvegicus channel protein, achieved by an in silico approach, is also presented.

Cryo-EM Structure of K+-Bound hERG Channel Complexed with the Blocker Astemizole.

The hERG channel is a voltage-gated potassium channel involved in cardiac repolarization. Off-target hERG inhibition by drugs has become a critical issue in the pharmaceutical industry. The three-dimensional structure of the hERG channel was recently reported at 3.8-Å resolution using cryogenic electron microscopy (cryo-EM). However, the drug inhibition mechanism remains unclear because of the scarce structural information regarding the drug- and potassium-bound hERG channels. In this study, we obtained the cryo-EM density map of potassium-bound hERG channel complexed with astemizole, a well-known hERG inhibitor that increases risk of potentially fatal arrhythmia, at 3.5-Å resolution. The structure suggested that astemizole inhibits potassium conduction by binding directly below the selectivity filter. Furthermore, we propose a possible binding model of astemizole to the hERG channel and provide insights into the unusual sensitivity of hERG to several drugs.

Toward in vivo-relevant hERG safety assessment and mitigation strategies based on relationships between non-equilibrium blocker binding, three-dimensional channel-blocker interactions, dynamic occupancy, dynamic exposure, and cellular arrhythmia.

The human ether-a-go-go-related voltage-gated cardiac ion channel (commonly known as hERG) conducts the rapid outward repolarizing potassium current in cardiomyocytes (IKr). Inadvertent blockade of this channel by drug-like molecules represents a key challenge in pharmaceutical R&D due to frequent overlap between the structure-activity relationships of hERG and many primary targets. Building on our previous work, together with recent cryo-EM structures of hERG, we set about to better understand the energetic and structural basis of promiscuous blocker-hERG binding in the context of Biodynamics theory. We propose a two-step blocker binding process consisting of: The initial capture step: diffusion of a single fully solvated blocker copy into a large cavity lined by the intra-cellular cyclic nucleotide binding homology domain (CNBHD). Occupation of this cavity is a necessary but insufficient condition for ion current disruption.The IKr disruption step: translocation of the captured blocker along the channel axis, such that: The head group, consisting of a quasi-rod-shaped moiety, projects into the open pore, accompanied by partial de-solvation of the binding interface.One tail moiety packs along a kink between the S6 helix and proximal C-linker helix adjacent to the intra-cellular entrance of the pore, likewise accompanied by mutual de-solvation of the binding interface (noting that the association barrier is comprised largely of the total head + tail group de-solvation cost).Blockers containing a highly planar moiety that projects into a putative constriction zone within the closed channel become trapped upon closing, as do blockers terminating prior to this region.A single captured blocker copy may conceivably associate and dissociate to/from the pore many times before exiting the CNBHD cavity. Lastly, we highlight possible flaws in the current hERG safety index (SI), and propose an alternate in vivo-relevant strategy factoring in: Benefit/risk.The predicted arrhythmogenic fractional hERG occupancy (based on action potential (AP) simulations of the undiseased human ventricular cardiomyocyte).Alteration of the safety threshold due to underlying disease.Risk of exposure escalation toward the predicted arrhythmic limit due to patient-to-patient pharmacokinetic (PK) variability, drug-drug interactions, overdose, and use for off-label indications in which the hERG safety parameters may differ from their on-label counterparts.

Evaluation of QSAR Equations for Virtual Screening.

Quantitative Structure Activity Relationship (QSAR) models can inform on the correlation between activities and structure-based molecular descriptors. This information is important for the understanding of the factors that govern molecular properties and for designing new compounds with favorable properties. Due to the large number of calculate-able descriptors and consequently, the much larger number of descriptors combinations, the derivation of QSAR models could be treated as an optimization problem. For continuous responses, metrics which are typically being optimized in this process are related to model performances on the training set, for example, R2 and QCV2. Similar metrics, calculated on an external set of data (e.g., QF1/F2/F32), are used to evaluate the performances of the final models. A common theme of these metrics is that they are context -" ignorant". In this work we propose that QSAR models should be evaluated based on their intended usage. More specifically, we argue that QSAR models developed for Virtual Screening (VS) should be derived and evaluated using a virtual screening-aware metric, e.g., an enrichment-based metric. To demonstrate this point, we have developed 21 Multiple Linear Regression (MLR) models for seven targets (three models per target), evaluated them first on validation sets and subsequently tested their performances on two additional test sets constructed to mimic small-scale virtual screening campaigns. As expected, we found no correlation between model performances evaluated by "classical" metrics, e.g., R2 and QF1/F2/F32 and the number of active compounds picked by the models from within a pool of random compounds. In particular, in some cases models with favorable R2 and/or QF1/F2/F32 values were unable to pick a single active compound from within the pool whereas in other cases, models with poor R2 and/or QF1/F2/F32 values performed well in the context of virtual screening. We also found no significant correlation between the number of active compounds correctly identified by the models in the training, validation and test sets. Next, we have developed a new algorithm for the derivation of MLR models by optimizing an enrichment-based metric and tested its performances on the same datasets. We found that the best models derived in this manner showed, in most cases, much more consistent results across the training, validation and test sets and outperformed the corresponding MLR models in most virtual screening tests. Finally, we demonstrated that when tested as binary classifiers, models derived for the same targets by the new algorithm outperformed Random Forest (RF) and Support Vector Machine (SVM)-based models across training/validation/test sets, in most cases. We attribute the better performances of the Enrichment Optimizer Algorithm (EOA) models in VS to better handling of inactive random compounds. Optimizing an enrichment-based metric is therefore a promising strategy for the derivation of QSAR models for classification and virtual screening.

Electrophysiological characterization of the modified hERGT potassium channel used to obtain the first cryo-EM hERG structure.

The voltage-gated hERG (human-Ether-à-go-go Related Gene) K+ channel plays a fundamental role in cardiac action potential repolarization. Loss-of-function mutations or pharmacological inhibition of hERG leads to long QT syndrome, whilst gain-of-function mutations lead to short QT syndrome. A recent open channel cryo-EM structure of hERG represents a significant advance in the ability to interrogate hERG channel structure-function. In order to suppress protein aggregation, a truncated channel construct of hERG (hERGT ) was used to obtain this structure. In hERGT cytoplasmic domain residues 141 to 350 and 871 to 1,005 were removed from the full-length channel protein. There are limited data on the electrophysiological properties of hERGT channels. Therefore, this study was undertaken to determine how hERGT influences channel function at physiological temperature. Whole-cell measurements of hERG current (IhERG ) were made at 37°C from HEK 293 cells expressing wild-type (WT) or hERGT channels. With a standard +20 mV activating command protocol, neither end-pulse nor tail IhERG density significantly differed between WT and hERGT . However, the IhERG deactivation rate was significantly slower for hERGT . Half-maximal activation voltage (V0.5 ) was positively shifted for hERGT by ~+8 mV (p < .05 versus WT), without significant change to the activation relation slope factor. Neither the voltage dependence of inactivation, nor time course of development of inactivation significantly differed between WT and hERGT , but recovery of IhERG from inactivation was accelerated for hERGT (p < .05 versus WT). Steady-state "window" current was positively shifted for hERGT with a modest increase in the window current peak. Under action potential (AP) voltage clamp, hERGT IhERG showed modestly increased current throughout the AP plateau phase with a significant increase in current integral during the AP. The observed consequences for hERGT IhERG of deletion of the two cytoplasmic regions may reflect changes to electrostatic interactions influencing the voltage sensor domain.

Long QT Syndrome Type 2: Emerging Strategies for Correcting Class 2 KCNH2 (hERG) Mutations and Identifying New Patients.

Significant advances in our understanding of the molecular mechanisms that cause congenital long QT syndrome (LQTS) have been made. A wide variety of experimental approaches, including heterologous expression of mutant ion channel proteins and the use of inducible pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from LQTS patients offer insights into etiology and new therapeutic strategies. This review briefly discusses the major molecular mechanisms underlying LQTS type 2 (LQT2), which is caused by loss-of-function (LOF) mutations in the KCNH2 gene (also known as the human ether-à-go-go-related gene or hERG). Almost half of suspected LQT2-causing mutations are missense mutations, and functional studies suggest that about 90% of these mutations disrupt the intracellular transport, or trafficking, of the KCNH2-encoded Kv11.1 channel protein to the cell surface membrane. In this review, we discuss emerging strategies that improve the trafficking and functional expression of trafficking-deficient LQT2 Kv11.1 channel proteins to the cell surface membrane and how new insights into the structure of the Kv11.1 channel protein will lead to computational approaches that identify which KCNH2 missense variants confer a high-risk for LQT2.

Serine mutation of a conserved threonine in the hERG K+ channel S6-pore region leads to loss-of-function through trafficking impairment.

The human Ether-à-go-go Related Gene (hERG) encodes a potassium channel responsible for the cardiac rapid delayed rectifier K+ current, IKr, which regulates ventricular repolarization. Loss-of-function hERG mutations underpin the LQT2 form of congenital long QT syndrome. This study was undertaken to elucidate the functional consequences of a variant of uncertain significance, T634S, located at a highly conserved position at the top of the S6 helix of the hERG channel. Whole-cell patch-clamp recordings were made at 37 °C of hERG current (IhERG) from HEK 293 cells expressing wild-type (WT) hERG, WT+T634S and hERG-T634S alone. When the T634S mutation was expressed alone little or no IhERG could be recorded. Co-expressing WT and hERG-T634S suppressed IhERG tails by ∼57% compared to WT alone, without significant alteration of voltage dependent activation of IhERG. A similar suppression of IhERG was observed under action potential voltage clamp. Comparable reduction of IKr in a ventricular AP model delayed repolarization and led to action potential prolongation. A LI-COR® based On/In-Cell Western assay showed that cell surface expression of hERG channels in HEK 293 cells was markedly reduced by the T634S mutation, whilst total cellular hERG expression was unaffected, demonstrating impaired trafficking of the hERG-T634S mutant. Incubation with E-4031, but not lumacaftor, rescued defective hERG-T634S channel trafficking and IhERG density. In conclusion, these data identify hERG-T634S as a rescuable trafficking defective mutation that reduces IKr sufficiently to delay repolarization and, thereby, potentially produce a LQT2 phenotype.

HERG channel and cancer: A mechanistic review of carcinogenic processes and therapeutic potential.

The human ether-à-go-go related gene (HERG) encodes the alpha subunit of Kv11.1, which is a voltage-gated K+ channel protein mainly expressed in heart and brain tissue. HERG plays critical role in cardiac repolarization, and mutations in HERG can cause long QT syndrome. More recently, evidence has emerged that HERG channels are aberrantly expressed in many kinds of cancer cells and play important roles in cancer progression. HERG could therefore be a potential biomarker for cancer and a possible molecular target for anticancer drug design. HERG affects a number of cellular processes, including cell proliferation, apoptosis, angiogenesis and migration, any of which could be affected by dysregulation of HERG. This review provides an overview of available information on HERG channel as it relates to cancer, with focus on the mechanism by which HERG influences cancer progression. Molecular docking attempts suggest two possible protein-protein interactions of HERG with the ß1-integrin receptor and the transcription factor STAT-1 as novel HERG-directed therapeutic targeting which avoids possible cardiotoxicity. The role of epigenetics in regulating HERG channel expression and activity in cancer will also be discussed. Finally, given its inherent extracellular accessibility as an ion channel, we discuss regulatory roles of this molecule in cancer physiology and therapeutic potential. Future research should be directed to explore the possibilities of therapeutic interventions targeting HERG channels while minding possible complications.