Selectivity filter modalities and rapid inactivation of the hERG1 channel.

The human ether-á-go-go-related gene (hERG1) channel conducts small outward K+ currents that are critical for cardiomyocyte membrane repolarization. The gain-of-function mutation N629D at the outer mouth of the selectivity filter (SF) disrupts inactivation and K+-selective transport in hERG1, leading to arrhythmogenic phenotypes associated with long-QT syndrome. Here, we combined computational electrophysiology with Markov state model analysis to investigate how SF-level gating modalities control selective cation transport in wild-type (WT) and mutant (N629D) hERG1 variants. Starting from the recently reported cryogenic electron microscopy (cryo-EM) open-state channel structure, multiple microseconds-long molecular-dynamics (MD) trajectories were generated using different cation configurations at the filter, voltages, electrolyte concentrations, and force-field parameters. Most of the K+ permeation events observed in hERG1-WT simulations occurred at microsecond timescales, influenced by the spontaneous dehydration/rehydration dynamics at the filter. The SF region displayed conductive, constricted, occluded, and dilated states, in qualitative agreement with the well-documented flickering conductance of hERG1. In line with mutagenesis studies, these gating modalities resulted from dynamic interaction networks involving residues from the SF, outer-mouth vestibule, P-helices, and S5-P segments. We found that N629D mutation significantly stabilizes the SF in a state that is permeable to both K+ and Na+, which is reminiscent of the SF in the nonselective bacterial NaK channel. Increasing the external K+ concentration induced "WT-like" SF dynamics in N629D, in qualitative agreement with the recovery of flickering currents in experiments. Overall, our findings provide an understanding of the molecular mechanisms controlling selective transport in K+ channels with a nonconventional SF sequence.

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.

Refinement of a cryo-EM structure of hERG: Bridging structure and function.

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.

Toward Reducing hERG Affinities for DAT Inhibitors with a Combined Machine Learning and Molecular Modeling Approach.

Psychostimulant drugs, such as cocaine, inhibit dopamine reuptake via blockading the dopamine transporter (DAT), which is the primary mechanism underpinning their abuse. Atypical DAT inhibitors are dissimilar to cocaine and can block cocaine- or methamphetamine-induced behaviors, supporting their development as part of a treatment regimen for psychostimulant use disorders. When developing these atypical DAT inhibitors as medications, it is necessary to avoid off-target binding that can produce unwanted side effects or toxicities. In particular, the blockade of a potassium channel, human ether-a-go-go (hERG), can lead to potentially lethal ventricular tachycardia. In this study, we established a counter screening platform for DAT and against hERG binding by combining machine learning-based quantitative structure-activity relationship (QSAR) modeling, experimental validation, and molecular modeling and simulations. Our results show that the available data are adequate to establish robust QSAR models, as validated by chemical synthesis and pharmacological evaluation of a validation set of DAT inhibitors. Furthermore, the QSAR models based on subsets of the data according to experimental approaches used have predictive power as well, which opens the door to target specific functional states of a protein. Complementarily, our molecular modeling and simulations identified the structural elements responsible for a pair of DAT inhibitors having opposite binding affinity trends at DAT and hERG, which can be leveraged for rational optimization of lead atypical DAT inhibitors with desired pharmacological properties.

The Pore-Lipid Interface: Role of Amino-Acid Determinants of Lipophilic Access by Ivabradine to the hERG1 Pore Domain.

Abnormal cardiac electrical activity is a common side effect caused by unintended block of the promiscuous drug target human ether-à-go-go-related gene (hERG1), the pore-forming domain of the delayed rectifier K+ channel in the heart. hERG1 block leads to a prolongation of the QT interval, a phase of the cardiac cycle that underlies myocyte repolarization detectable on the electrocardiogram. Even newly released drugs such as heart-rate lowering agent ivabradine block the rapid delayed rectifier current IKr, prolong action potential duration, and induce potentially lethal arrhythmia known as torsades de pointes. In this study, we describe a critical drug-binding pocket located at the lateral pore surface facing the cellular membrane. Mutations of the conserved M651 residue alter ivabradine-induced block but not by the common hERG1 blocker dofetilide. As revealed by molecular dynamics simulations, binding of ivabradine to a lipophilic pore access site is coupled to a state-dependent reorientation of aromatic residues F557 and F656 in the S5 and S6 helices. We show that the M651 mutation impedes state-dependent dynamics of F557 and F656 aromatic cassettes at the protein-lipid interface, which has a potential to disrupt drug-induced block of the channel. This fundamentally new mechanism coupling the channel dynamics and small-molecule access from the membrane into the hERG1 intracavitary site provides a simple rationale for the well established state-dependence of drug blockade. SIGNIFICANCE STATEMENT: The drug interference with the function of the cardiac hERG channels represents one of the major sources of drug-induced heart disturbances. We found a novel and a critical drug-binding pocket adjacent to a lipid-facing surface of the hERG1 channel, which furthers our molecular understanding of drug-induced QT syndrome.

Kinetic model for NS1643 drug activation of WT and L529I variants of Kv11.1 (hERG1) potassium channel.

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing IKr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.

NS1643 interacts around L529 of hERG to alter voltage sensor movement on the path to activation.

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.

Ivabradine prolongs phase 3 of cardiac repolarization and blocks the hERG1 (KCNH2) current over a concentration-range overlapping with that required to block HCN4.

In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the If current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance.

Phospholamban knockout breaks arrhythmogenic Ca²âº waves and suppresses catecholaminergic polymorphic ventricular tachycardia in mice.

RATIONALE: Phospholamban (PLN) is an inhibitor of cardiac sarco(endo)plasmic reticulum Ca²âº ATPase. PLN knockout (PLN-KO) enhances sarcoplasmic reticulum Ca²âº load and Ca²âº leak. Conversely, PLN-KO accelerates Ca²âº sequestration and aborts arrhythmogenic spontaneous Ca²âº waves (SCWs). An important question is whether these seemingly paradoxical effects of PLN-KO exacerbate or protect against Ca²âº-triggered arrhythmias. OBJECTIVE: We investigate the impact of PLN-KO on SCWs, triggered activities, and stress-induced ventricular tachyarrhythmias (VTs) in a mouse model of cardiac ryanodine-receptor (RyR2)-linked catecholaminergic polymorphic VT. METHODS AND RESULTS: We generated a PLN-deficient, RyR2-mutant mouse model (PLN-/-/RyR2-R4496C+/-) by crossbreeding PLN-KO mice with catecholaminergic polymorphic VT-associated RyR2-R4496C mutant mice. Ca²âº imaging and patch-clamp recording revealed cell-wide propagating SCWs and triggered activities in RyR2-R4496C+/- ventricular myocytes during sarcoplasmic reticulum Ca²âº overload. PLN-KO fragmented these cell-wide SCWs into mini-waves and Ca²âº sparks and suppressed the triggered activities evoked by sarcoplasmic reticulum Ca²âº overload. Importantly, these effects of PLN-KO were reverted by partially inhibiting sarco(endo)plasmic reticulum Ca²âº ATPase with 2,5-di-tert-butylhydroquinone. However, Bay K, caffeine, or Li⁺ failed to convert mini-waves to cell-wide SCWs in PLN-/-/RyR2-R4496C+/- ventricular myocytes. Furthermore, ECG analysis showed that PLN-KO mice are not susceptible to stress-induced VTs. On the contrary, PLN-KO protected RyR2-R4496C mutant mice from stress-induced VTs. CONCLUSIONS: Our results demonstrate that despite severe sarcoplasmic reticulum Ca²âº leak, PLN-KO suppresses triggered activities and stress-induced VTs in a mouse model of catecholaminergic polymorphic VT. These data suggest that breaking up cell-wide propagating SCWs by enhancing Ca²âº sequestration represents an effective approach for suppressing Ca²âº-triggered arrhythmias.

Series of (([1,1'-Biphenyl]-2-yl)methyl)sulfinylalkyl Alicyclic Amines as Novel and High Affinity Atypical Dopamine Transporter Inhibitors with Reduced hERG Activity.

Currently, there are no FDA-approved medications for the treatment of psychostimulant use disorders (PSUD). We have previously discovered "atypical" dopamine transporter (DAT) inhibitors that do not display psychostimulant-like behaviors and may be useful as medications to treat PSUD. Lead candidates (e.g., JJC8-091, 1) have shown promising in vivo profiles in rodents; however, reducing hERG (human ether-à-go-go-related gene) activity, a predictor of cardiotoxicity, has remained a challenge. Herein, a series of 30 (([1,1'-biphenyl]-2-yl)methyl)sulfinylalkyl alicyclic amines was synthesized and evaluated for DAT and serotonin transporter (SERT) binding affinities. A subset of analogues was tested for hERG activity, and the IC50 values were compared to those predicted by our hERG QSAR models, which showed robust predictive power. Multiparameter optimization scores (MPO > 3) indicated central nervous system (CNS) penetrability. Finally, comparison of affinities in human DAT and its Y156F and Y335A mutants suggested that several compounds prefer an inward facing conformation indicating an atypical DAT inhibitor profile.

The Nlrp3 inflammasome promotes myocardial dysfunction in structural cardiomyopathy through interleukin-1ß.

Heart failure is associated with a low-grade and chronic cardiac inflammation that impairs function; however, the mechanisms by which this sterile inflammation occurs in structural heart disease remain poorly defined. Cardiac-specific heterozygous overexpression of the calcineurin transgene (CNTg) in mice results in cardiac hypertrophy, inflammation, apoptosis and ventricular dilatation. We hypothesized that activation of the Nlrp3 inflammasome, an intracellular danger-sensing pathway required for processing the pro-inflammatory cytokine interleukin-1ß (IL-1ß), may contribute to myocardial dysfunction and disease progression. Here we report that Nlrp3 mRNA was increased in CNTg mice compared with wild-type. Consistent with inflammasome activation, CNTg animals had increased conversion of pro-caspase-1 to cleaved and activated forms, as well as markedly increased serum IL-1ß. Blockade of IL-1ß signalling via chronic IL-1 receptor antagonist therapy reduced cardiac inflammation and myocyte pathology in CNTg mice, resulting in improved systolic performance. Furthermore, genetic ablation of Nlrp3 in CNTg mice reduced pro-inflammatory cytokine maturation and cardiac inflammation, as well as improving systolic performance. These findings indicate that activation of the Nlrp3 inflammasome in CNTg mice promotes myocardial inflammation and systolic dysfunction through the production of pro-inflammatory IL-1ß. Blockade of IL-1ß signalling with the IL-1 receptor antagonist reverses these phenotypes and offers a possible therapeutic approach in the management of heart failure.

Structure-guided topographic mapping and mutagenesis to elucidate binding sites for the human ether-a-go-go-related gene 1 potassium channel (KCNH2) activator NS1643.

Loss-of -function mutations in human ether-a-go-go-related gene 1 (hERG1) is associated with life-threatening arrhythmias. hERG1 activators are being developed as treatments for acquired or genetic forms of long QT syndrome. The locations of the putative binding pockets for activators are still being elucidated. In silico docking of the activator 1,3-bis-(2-hydroxy-5-trifluoromethylphenyl)-urea (NS1643) to an S1-S6 transmembrane homology model of hERG1 predicted putative binding sites. The predictions of the in silico docking guided subsequent in vitro mutagenesis and electrophysiological measurements. The novel interacting site for NS1643 is predicted around Asn629 at the outer mouth of the channel. The applied N629H mutation is the sole amino acid replacement in the literature that abrogates the NS1643-induced left shift of the V(1/2) of activation. In contrast, both N629T and N629D showed pharmacologic responses similar to wild type. Another important interacting pocket is predicted at the intracellular surface in the S4-S5 linker. Mutagenesis of the residues critical to interactions in this pocket had major effects on the pharmacologic response to NS1643. The inward conductance elicited by hyperpolarization of D540K hERG1 was abrogated by NS1643 treatment, suggesting that it alters the inward movement of the S4 segment. The neighboring E544L mutation markedly exaggerated tail-current responses to NS1643. However, an L564A substitution inhibited drug response. Structure-guided mutagenesis identified widespread clusters of amino acids modulating drug-induced shifts in inactivation; such modulation may reflect allosteric changes in tertiary structure. Model-guided mutagenesis led to the discovery of a range of novel interacting residues that modify NS1643-induced pharmacologic responses.

Lipid regulation of hERG1 channel function.

The lipid regulation of mammalian ion channel function has emerged as a fundamental mechanism in the control of electrical signalling and transport specificity in various cell types. In this work, we combine molecular dynamics simulations, mutagenesis, and electrophysiology to provide mechanistic insights into how lipophilic molecules (ceramide-sphingolipid probe) alter gating kinetics and K+ currents of hERG1. We show that the sphingolipid probe induced a significant left shift of activation voltage, faster deactivation rates, and current blockade comparable to traditional hERG1 blockers. Microseconds-long MD simulations followed by experimental mutagenesis elucidated ceramide specific binding locations at the interface between the pore and voltage sensing domains. This region constitutes a unique crevice present in mammalian channels with a non-swapped topology. The combined experimental and simulation data provide evidence for ceramide-induced allosteric modulation of the channel by a conformational selection mechanism.

Structural refinement of the hERG1 pore and voltage-sensing domains with ROSETTA-membrane and molecular dynamics simulations.

The hERG1 gene (Kv11.1) encodes a voltage-gated potassium channel. Mutations in this gene lead to one form of the Long QT Syndrome (LQTS) in humans. Promiscuous binding of drugs to hERG1 is known to alter the structure/function of the channel leading to an acquired form of the LQTS. Expectably, creation and validation of reliable 3D model of the channel have been a key target in molecular cardiology and pharmacology for the last decade. Although many models were built, they all were limited to pore domain. In this work, a full model of the hERG1 channel is developed which includes all transmembrane segments. We tested a template-driven de-novo design with ROSETTA-membrane modeling using side-chain placements optimized by subsequent molecular dynamics (MD) simulations. Although backbone templates for the homology modeled parts of the pore and voltage sensors were based on the available structures of KvAP, Kv1.2 and Kv1.2-Kv2.1 chimera channels, the missing parts are modeled de-novo. The impact of several alignments on the structure of the S4 helix in the voltage-sensing domain was also tested. Herein, final models are evaluated for consistency to the reported structural elements discovered mainly on the basis of mutagenesis and electrophysiology. These structural elements include salt bridges and close contacts in the voltage-sensor domain; and the topology of the extracellular S5-pore linker compared with that established by toxin foot-printing and nuclear magnetic resonance studies. Implications of the refined hERG1 model to binding of blockers and channels activators (potent new ligands for channel activations) are discussed.

Allosteric Coupling Between Drug Binding and the Aromatic Cassette in the Pore Domain of the hERG1 Channel: Implications for a State-Dependent Blockade.

Human-ether-a-go-go-related channel (hERG1) is the pore-forming domain of the delayed rectifier K+ channel in the heart which underlies the IKr current. The channel has been extensively studied due to its propensity to bind chemically diverse group of drugs. The subsequent hERG1 block can lead to a prolongation of the QT interval potentially leading to an abnormal cardiac electrical activity. The recently solved cryo-EM structure featured a striking non-swapped topology of the Voltage-Sensor Domain (VSD) which is packed against the pore-domain as well as a small and hydrophobic intra-cavity space. The small size and hydrophobicity of the cavity was unexpected and challenges the already-established hypothesis of drugs binding to the wide cavity. Recently, we showed that an amphipathic drug, ivabradine, may favorably bind the channel from the lipid-facing surface and we discovered a mutant (M651T) on the lipid facing domain between the VSD and the PD which inhibited the blocking capacity of the drug. Using multi-microseconds Molecular Dynamics (MD) simulations of wild-type and M651T mutant hERG1, we suggested the block of the channel through the lipid mediated pathway, the opening of which is facilitated by the flexible phenylalanine ring (F656). In this study, we characterize the dynamic interaction of the methionine-aromatic cassette in the S5-S6 helices by combining data from electrophysiological experiments with MD simulations and molecular docking to elucidate the complex allosteric coupling between drug binding to lipid-facing and intra-cavity sites and aromatic cassette dynamics. We investigated two well-established hERG1 blockers (ivabradine and dofetilide) for M651 sensitivity through electrophysiology and mutagenesis techniques. Our electrophysiology data reveal insensitivity of dofetilide to the mutations at site M651 on the lipid facing side of the channel, mirroring our results obtained from docking experiments. Moreover, we show that the dofetilide-induced block of hERG1 occurs through the intracellular space, whereas little to no block of ivabradine is observed during the intracellular application of the drug. The dynamic conformational rearrangement of the F656 appears to regulate the translocation of ivabradine into the central cavity. M651T mutation appears to disrupt this entry pathway by altering the molecular conformation of F656.

Interactions of H562 in the S5 helix with T618 and S621 in the pore helix are important determinants of hERG1 potassium channel structure and function.

hERG1 is a member of the cyclic nucleotide binding domain family of K(+) channels. Alignment of cyclic nucleotide binding domain channels revealed an evolutionary conserved sequence HwX(A/G)C in the S5 domain. We reasoned that histidine 562 in hERG1 could play an important structure-function role. To explore this role, we created in silica models of the hERG1 pore domain based on the KvAP crystal structure with Rosetta-membrane modeling and molecular-dynamics simulations. Simulations indicate that the H562 residue in the S5 helix spans the gap between the S5 helix and the pore helix, stabilizing the pore domain, and that mutation at the H562 residue leads to a disruption of the hydrogen bonding to T618 and S621, resulting in distortion of the selectivity filter. Analysis of the simulated point mutations at positions 562/618/621 showed that the reciprocal double mutations H562W/T618I would partially restore the orientation of the 562 residue. Matching hydrophobic interactions between mutated W562 residue and I618 partially compensate for the disrupted hydrogen bonding. Complementary in vitro electrophysiological studies confirmed the results of the molecular-dynamics simulations on single mutations at positions 562, 618, and 621. Experimentally, mutations of the H562 to tryptophan produced a functional channel, but with slowed deactivation and shifted V(1/2) of activation. Furthermore, the double mutation T618I/H562W rescued the defects seen in activation, deactivation, and potassium selectivity seen with the H562W mutation. In conclusion, interactions between H562 in the S5 helix and amino acids in the pore helix are important determinants of hERG1 potassium channel function, as confirmed by theory and experiment.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study.

IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

Selective knockout of mouse ERG1 B potassium channel eliminates I(Kr) in adult ventricular myocytes and elicits episodes of abrupt sinus bradycardia.

The ERG1 gene encodes a family of potassium channels. Mutations in human ERG1 lead to defects in cardiac repolarization, referred to as the long QT syndrome. Through homologous recombination in mouse embryonic stem cells the ERG1 B potassium channel transcript was eliminated while the ERG1 A transcript was maintained. Heterologous expression of ERG1 isoforms had previously indicated that the deactivation time course of ERG1 B is 10-fold more rapid than that of ERG1 A. In day-18 fetal +/+ myocytes, I(Kr) exhibited two time constants of deactivation (3,933 +/- 404 and 350 +/- 19 ms at -50 mV), whereas in age-matched ERG1 B(-/-) mice the rapid component was absent. Biexponential deactivation rates (2,039 +/- 268 and 163 +/- 43 ms at -50 mV) were also observed in adult +/+ myocytes. In adult ERG1 B(-/-) myocytes no I(Kr) was detected. Electrocardiogram intervals were similar in +/+ and -/- mice. However, adult -/- mice manifested abrupt spontaneous episodes of sinus bradycardia (>100 ms of slowing) in 6 out of 21 mice. This phenomenon was never observed in +/+ mice (0 out of 16). We conclude that ERG1 B is necessary for I(Kr) expression in the surface membrane of adult myocytes. Knockout of ERG1 B predisposes mice to episodic sinus bradycardia.