hERG Gating Research Articles

Determining the role of the unique N-terminal 1b domain in the gating of heterotetrameric hERG1a/hERG1b potassium channels.

Two isoforms of hERG (human Ether-a-go-go-Related Gene) potassium channel, hERG1a and hERG1b, associate in the heart to form the rapid delayed rectifier current (IKr), which is primarily responsible for repolarizing cardiac action potentials. Inherited mutations in both hERG1a and hERG1b cause Long QT Syndrome type 2 (LQT2), marked by prolonged cardiac repolarization and sudden death arrhythmia. The assembly of hERG1a subunits with hERG1b subunits increases the number of hERG1b subunits at the plasma membrane, but the mechanism by which hERG1a enhances hERG1b is not completely understood. Recently, it was reported that when expressed in trans, the hERG1a N-terminal region markedly increased hERG1b currents and increased biotin-labelled hERG1b protein at the plasma membrane surface. A direct structural interaction between the hERG1a N-terminal residues 216-220 and the hERG1b N-terminal region was characterized and proposed to be necessary for the upregulation of hERG1b (Johnson, Crawford, and Trudeau, 2022). However, hERG1b channels with a deletion of the unique N-terminal 1b domain did not have a measurable increase in current density or in biotinylated protein when co-expressed with hERG1a N-terminal region pieces. This indicates that the 1b domain is required for the increase in hERG1b by hERG1a. Interestingly, the 1b domain-deleted hERG1b channels displayed a large leftward shift in the voltage dependence of activation when co-expressed with the full hERG1a N-terminal region. This is unexpected, as the 1b domain was not previously considered as a determinant of hERG gating. Here, we will investigate the mechanism of this large leftward shift and determine the role of the 1b domain in the gating properties of hERG1a/hERG1b heterotetrameric channels.

Hysteretic hERG channel gating current recorded at physiological temperature

Cardiac hERG channels comprise at least two subunits, hERG 1a and hERG 1b, and drive cardiac action potential repolarization. hERG 1a subunits contain a cytoplasmic PAS domain that is absent in hERG 1b. The hERG 1a PAS domain regulates voltage sensor domain (VSD) movement, but hERG VSD behavior and its regulation by the hERG 1a PAS domain have not been studied at physiological temperatures. We recorded gating charge from homomeric hERG 1a and heteromeric hERG 1a/1b channels at near physiological temperatures (36 ± 1 °C) using pulse durations comparable in length to the human ventricular action potential. The voltage dependence of deactivation was hyperpolarized relative to activation, reflecting VSD relaxation at positive potentials. These data suggest that relaxation (hysteresis) works to delay pore closure during repolarization. Interestingly, hERG 1a VSD deactivation displayed a double Boltzmann distribution, but hERG 1a/1b deactivation displayed a single Boltzmann. Disabling the hERG 1a PAS domain using a PAS-targeting antibody similarly transformed hERG 1a deactivation from a double to a single Boltzmann, highlighting the contribution of the PAS in regulating VSD movement. These data represent, to our knowledge, the first recordings of hERG gating charge at physiological temperature and demonstrate that VSD relaxation (hysteresis) is present in hERG channels at physiological temperature.

Effects of Antiarrhythmic Drugs on hERG Gating in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes From a Patient With Short QT Syndrome Type 1.

Aims: The short QT syndrome type 1 (SQT1) is linked to hERG channel mutations (e.g., N588K). Drug effects on hERG channel gating kinetics in SQT1-cells have not been investigated. Methods: This study used hiPSC-CMs of a healthy donor and a SQT1-patient carrying the N588K mutation and patch clamp to examine the drug effects on hERG channel gating kinetics. Results: Ajmaline, amiodarone, ivabradine, flecainide, quinidine, mexiletine and ranolazine inhibited the hERG channel current (IKr) less strongly in hiPSC-CMs from the SQTS1-patient (SQT1-hiPSC-CMs) comparing with cells from the healthy donor (donor-hiPSC-CMs). Quinidine and mexiletine reduced, but ajmaline, amiodarone, ivabradine and ranolazine increased the time to peak of IKr similarly in SQT1-hiPSC-CMs and donor-hiPSC-CMs. Although regarding the shift of activation and inactivation curves, tested drugs showed differential effects in donor- and SQT1-hiPSC-CMs, quinidine, ajmaline, ivabradine and mexiletine but not amiodarone, flecainide and ranolazine reduced the window current in SQT1-hiPSC-CMs. Quinidine, ajmaline, ivabradine and mexiletine differentially changed the time constant of recovery from inactivation, but all of them increased the time constant of deactivation in SQT1-hiPSC-CMs. Conclusion: The window current-reducing and deactivation-slowing effects may be important for the antiarrhythmic effect of ajmaline, ivabradine, quinidine and mexiletine in SQT1-cells. This information may be helpful for selecting drugs for treating SQT1-patients with hERG channel mutation.

Superparamagnetic iron oxide nanoparticles (SPIONs) modulate hERG ion channel activity

Superparamagnetic iron oxide nanoparticles (SPIONs) are widely used in various biomedical applications, such as diagnostic agents in magnetic resonance imaging (MRI), for drug delivery vehicles and in hyperthermia treatment of tumors. Although the potential benefits of SPIONs are considerable, there is a distinct need to identify any potential cellular damage associated with their use. Since human ether à go-go-related gene (hERG) channel, a protein involved in the repolarization phase of cardiac action potential, is considered one of the main targets in the drug discovery process, we decided to evaluate the effects of SPIONs on hERG channel activity and to determine whether the oxidation state, the dimensions and the coating of nanoparticles (NPs) can influence the interaction with hERG channel. Using patch clamp recordings, we found that SPIONs inhibit hERG current and this effect depends on the coating of NPs. In particular, SPIONs with covalent coating aminopropylphosphonic acid (APPA) have a milder effect on hERG activity. We observed that the time-course of hERG channel modulation by SPIONs is biphasic, with a transient increase (∼20% of the amplitude) occurring within the first 1–3 min of perfusion of NPs, followed by a slower inhibition. Moreover, in the presence of SPIONs, deactivation kinetics accelerated and the activation and inactivation I–V curves were right-shifted, similarly to the effect described for the binding of other divalent metal ions (e.g. Cd2+ and Zn2+). Finally, our data show that a bigger size and the complete oxidation of SPIONs can significantly decrease hERG channel inhibition. Taken together, these results support the view that Fe2+ ions released from magnetite NPs may represent a cardiac risk factor, since they alter hERG gating and these alterations could compromise the cardiac action potential.

Rapid Characterization of hERG Channel Kinetics II: Temperature Dependence

Ion channel behavior can depend strongly on temperature, with faster kinetics at physiological temperatures leading to considerable changes in currents relative to room temperature. These temperature-dependent changes in voltage-dependent ion channel kinetics (rates of opening, closing, inactivating, and recovery) are commonly represented with Q10 coefficients or an Eyring relationship. In this article, we assess the validity of these representations by characterizing channel kinetics at multiple temperatures. We focus on the human Ether-à-go-go-Related Gene (hERG) channel, which is important in drug safety assessment and commonly screened at room temperature so that results require extrapolation to physiological temperature. In Part I of this study, we established a reliable method for high-throughput characterization of hERG1a (Kv11.1) kinetics, using a 15-second information-rich optimized protocol. In this Part II, we use this protocol to study the temperature dependence of hERG kinetics using Chinese hamster ovary cells overexpressing hERG1a on the Nanion SyncroPatch 384PE, a 384-well automated patch-clamp platform, with temperature control. We characterize the temperature dependence of hERG gating by fitting the parameters of a mathematical model of hERG kinetics to data obtained at five distinct temperatures between 25 and 37°C and validate the models using different protocols. Our models reveal that activation is far more temperature sensitive than inactivation, and we observe that the temperature dependency of the kinetic parameters is not represented well by Q10 coefficients; it broadly follows a generalized, but not the standardly-used, Eyring relationship. We also demonstrate that experimental estimations of Q10 coefficients are protocol dependent. Our results show that a direct fit using our 15-s protocol best represents hERG kinetics at any given temperature and suggests that using the Generalized Eyring theory is preferable if no experimental data are available to derive model parameters at a given temperature.

Conformational Dynamics of the Intrinsic Ligand in the CNBHD of the Voltage-Gated Potassium Channel hERG

Human ether-á-go-go related gene (hERG) voltage-activated potassium channels are critical for cardiac excitability. Characteristic slow closing (deactivation) in hERG is regulated by a direct interaction between the N-terminal Per-Arnt-Sim (PAS) domain and the C-terminal cyclic nucleotide binding homology domain (CNBHD). An intrinsic ligand in hERG is located at the PAS-CNBHD interface, but its mechanism is not well understood. Our recent work with co-expressed channel fragments in which the hERG PAS domain fused to CFP was expressed in trans with the ‘core’ of the hERG channel (i.e. a channel lacking the PAS domain that is fused to Citrine at the C-terminus, hERG ΔPAS-Citrine) showed that deactivation gating was disrupted (was made faster) by intrinsic ligand mutations and that the activation time course of the channel was perturbed (was made faster), indicating that the intrinsic ligand is necessary for the functional and structural interaction of the PAS domain and the CNBHD. To better understand the dynamics for intrinsic ligand regulation of hERG gating, we incorporated the fluorescent non-canonical amino acid ANAP using amber codon suppression at the intrinsic ligand, and engineered dihistidines that were structurally adjacent to this ANAP, to allow us to probe a change (1) using an environmentally-dependent spectral shift of ANAP and/or (2) using transition metal FRET. This technique will determine how the intrinsic ligand at the PAS-CNBHD interface may undergo conformational changes with voltage to regulate channel deactivation gating.

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.

The Fast Component of hERG Gating Charge: An Interaction between D411 in the S1 and S4 Residues

Kv11.1 (hERG) is a voltage-gated potassium channel that shows very slow ionic current activation kinetics, and an unusual underlying biphasic gating charge movement with fast and slow components that differ greatly in time course. The structural basis and role of the fast component of gating charge (Qfast) is unclear, and its relationship to the slow activation of hERG channels is not understood. In this study we have used the cut-open oocyte voltage-clamp technique to investigate the relationship of fast gating charge movement-to-residue interactions between D411 at the bottom of the S1, and lower S4 domain charged and uncharged residues. Neutralization of D411 or K538 and V535A prevented Qfast and greatly accelerated overall charge movement. Voltage-clamp fluorometry showed a loss of a fast component of S4 fluorescence in D411N, V535A, and K538Q upon depolarization, whereas [2-(trimethyl ammonium) ethyl] methanethiosulfonate chloride modification of I521C in the outer S4 was enhanced at more negative potentials and at earlier times in these same mutants. A functional interaction between these regions during activation was suggested by ΔΔGo values >4.2 kJ/mol obtained from double mutant cycle analysis. The data indicate that interactions of S1 residue D411 with lower S4 residues stabilizes early closed states of the channel, and that disruption of these interactions results in both faster rates of activation gating and an elimination of the fast component of gating charge movement and of fluorescence. We propose that the Qfast charge movement during activation accompanies transitions through early closed states of the hERG activation pathway, and that the weak voltage dependence of these transitions limits the overall activation rate of hERG channels. Disruption of the D411-S4 interactions destabilizes these early closed states, leaving hERG channels able to activate at a rate similar to conventional potassium channels.

A temperature-dependent in silico model of the human ether-à-go-go-related (hERG) gene channel

IntroductionCurrent regulatory guidelines for assessing the risk of QT prolongation include in vitro assays assessing drug effects on the human ether-à-go-go-related (hERG; also known as Kv11.1) channel expressed in cell lines. These assays are typically conducted at room temperature to promote the ease and stability of recording hERG currents. However, the new Comprehensive in vitro Proarrhythmia Assay (CiPA) paradigm proposes to use an in silico model of the human ventricular myocyte to assess risk, requiring as input hERG channel pharmacology data obtained at physiological temperatures. To accommodate current industry safety pharmacology practices for measuring hERG channel activity, an in silico model of hERG channel that allows for the extrapolation of hERG assay data across different temperatures is desired. Because temperature may have an effect on both channel gating and drug binding rate, such models may need to have two components: a base model dealing with temperature-dependent gating changes without drug, and a pharmacodynamic component simulating temperature-dependent drug binding kinetics. As a first step, a base mode that can capture temperature effects on hERG channel gating without drug is needed. Methods and resultsTo meet this need for a temperature-dependent base model, a Markov model of the hERG channel with state transition rates explicitly dependent on temperature was developed and calibrated using data from a variety of published experiments conducted over a range of temperatures. The model was able to reproduce observed temperature-dependent changes in key channel gating properties and also to predict the results obtained in independent sets of new experiments. DiscussionThis new temperature-sensitive model of hERG gating represents an attempt to improve the predictivity of safety pharmacology testing by enabling the translation of room temperature hERG assay data to more physiological conditions. With further development, this model can be incorporated into the CiPA paradigm and also be used as a tool for developing insights into the thermodynamics of hERG channel gating mechanisms and the temperature-dependence of hERG channel block by drugs.

Getting to the heart of hERG K(+) channel gating.

Potassium ion channels encoded by the human ether-a-go-go related gene (hERG) form the ion-conducting subunit of the rapid delayed rectifier potassium current (IKr ). Although hERG channels exhibit a widespread tissue distribution they play a particularly important role in the heart. There has been considerable interest in hERG K(+) channels for three main reasons. First, they have very unusual gating kinetics, most notably rapid and voltage-dependent inactivation coupled to slow deactivation, which has led to the suggestion that they may play a specific role in the suppression of arrhythmias. Second, mutations in hERG are the cause of 30-40% of cases of congenital long QT syndrome (LQTS), the commonest inherited primary arrhythmia syndrome. Third, hERG is the molecular target for the vast majority of drugs that cause drug-induced LQTS, the commonest cause of drug-induced arrhythmias and cardiac death. Drug-induced LQTS has now been reported for a large range of both cardiac and non-cardiac drugs, in which this side effect is entirely undesired. In recent years there have been comprehensive reviews published on hERG K(+) channels (Vandenberg etal. 2012) and we will not re-cover this ground. Rather, we focus on more recent work on the structural basis and dynamics of hERG gating with an emphasis on how the latest developments may facilitate translational research in the area of stratifying risk of arrhythmias.

An Eag Domain Polypeptide Regulates the Deactivation Kinetics of the hERG 1A-3.1 Splice Variant Linked to Schizophrenia

The Human Ether-a-go-go Related Gene (hERG) encodes a voltage-activated K+ channel. hERG contributes to the repolarization of the ventricular action potential as the primary component of the cardiac delayed rectifier K+ current (IKr) and has also been shown by many groups to modulate neuronal firing frequency. Disruption of channel function by inherited mutations in the gene encoding hERG has been shown to cause type 2 long QT syndrome (LQT2). hERG gating is characterized by rapid inactivation upon depolarization and rapid recovery from inactivation and slow closing (deactivation) upon repolarization. Unlike other K+ channels, hERG, ELK and EAG channels contain an N-terminal eag domain, composed of a PAS domain and a PAS-Cap domain in the N-terminal region. Inter-subunit interactions between the N-terminal eag domain and the C-terminal cyclic-nucleotide binding homology domain regulate slow deactivation kinetics in the original variant of hERG, hERG1a channels. A recently discovered splice variant, hERG 1a-3.1, has much faster deactivation and has been linked to schizophrenia. We demonstrate in HEK293 cells, that co-expression of hERG1a-3.1 with soluble eag domain peptide partially rescues deactivation kinetics, presumably by providing a fully functional eag domain to the eag-truncated hERG1a-3.1 to slow channel deactivation. Cells co-expressing hERG1a-3.1 + eag domain polypeptides had significantly slower fast and slow time constants of deactivation compared to hERG1a-3.1 channels alone (p<0.05). We conclude that eag domain polypeptides are able to partially restore deactivation gating. Current experiments are utilizing FRET to assess if the hERG1a-3.1 C-terminus interaction with the eag domain is physically disrupted.

HERG Channel Voltage Sensor Movement Precedes Pore Opening

hERG (Kv11.1) voltage gated potassium channels are notable for their slow activation, fast inactivation and slow deactivation. The unique kinetics of hERG gating tunes the channel activity to provide a vital repolarizing potassium current during the late stage of the plateau of the action potential that sets its duration. Unlike most Kv1 channels which show rapid gating charge movement that kinetically precedes ionic current activation, the bulk of charge movement in hERG is slow. This suggests that the rate of hERG channel opening might be determined by slow voltage sensor movement, but no detailed kinetic analysis of gating charge movement has yet been reported. The fundamental question of whether voltage sensor movement or a weak coupling mechanism is responsible for slow pore activation therefore remains unresolved. To clarify the relationship between voltage sensor domain rearrangement and pore opening we performed a kinetic analysis of ionic current activation and voltage sensor movement using the Cut Open Oocyte Clamp technique. At 0 mV, close to the V0.5 for channel conductance, gating charge moved ∼3-fold faster than ionic currents with time constants τ=88 ms and τ=290 ms for gating and ionic activation respectively. At +60 mV, gating charge moved with a time constant τ=30 ms, also faster than the ionic current activation time constant τ=42 ms. These data indicate that the gating mechanism in hERG channels that underlies slow activation is not solely dependent upon the bulk of charge movement and suggests the presence of additional rate limiting transitions downstream of charge movements in the activation pathway.

External protons destabilize the activated voltage sensor in hERG channels

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium

The human ether-a-go-go related (hERG) potassium channel has unusual functional characteristics in that the rates of channel activation and deactivation are much slower than inactivation, which is attributed to specific structural elements within the NH2 terminus and the S1-S4 voltage-sensing domains (VSD). Although the charged residues in the VSD have been extensively modified and mutated as a result, the role and importance of specific hydrophobic residues in the S4 has been much less explored in studies of hERG gating. We found that charged, but not neutral or hydrophobic, amino acid substitution of isoleucine 521 at the outer end of the S4 transmembrane domain resulted in channels activating at much more negative voltages associated with a marked hyperpolarization of the conductance-voltage (G-V) relationship. The contributions of different physicochemical properties to this effect were probed by chemical modification of channels substituted with cysteine at position I521. When positively charged reagents including tetramethyl-rhodamine-5-maleimide (TMRM), 1-(2-maleimidylethyl)-4-[5-(4-methoxyphenyl)oxazol-2-yl] pyridinium methane-sulfonate (PyMPO), [2-(trimethylammonium)ethyl] methanethiosulfonate chloride (MTSET), and 2-aminoethyl methanethiosulfonate hydrobromide (MTSEA) were bound to the cysteine, I521C channels activated at more negative membrane potentials. To examine the contributions to hERG gating of other residues at the outer end of S4 (520-528), we performed a cysteine scan combined with MTSET modification. Only L520C, along with I521C, shows a substantial hyperpolarizing shift of the G-V relationship upon MTSET modification. The data indicate that the neutral, hydrophobic residue I521 at the extracellular end of S4 is critical for stabilizing the closed conformation of the hERG channel relative to the open state and by comparison with Shaker supports the alignment of hERG I521 with Shaker L361.

HERG Activation Gating Is Rapid: Evidence from Gating-Current Recordings and MTSET Modification of the Voltage Sensor

Background The human ether-a-go-go related gene protein (hERG) K + channel (Kv11.1) plays a major role in cardiac action potential repolarization, and defects in its function/expression can lead to long QT syndrome. The channel's key physiologic function as the main repolarization current is attributable in part to its unconventional slow activation kinetics, which are an order of magnitude slower than other members of the voltage-gated potassium channel superfamily. Methods We recorded gating-currents of hERG channels in tsa201 cells in order to directly identify the rate-limiting step in the activation of hERG channels. Expression of hERG channels was optimized by subcloning channel cDNA into a pGW1H expression vector and transfection into tsa201 cells. Gating currents were recorded in NMG + -based potassium-free internal and external solutions. As well, we used MTS modification of an externally facing residue in S4, I521C, to track the time course of movement of the S4 voltage sensor. I521 was mutated to a cysteine residue expressed in Xenopus or tsa201 cells, and various thiol-modifying reagents were used to modulate hERG activation and deactivation kinetics. Results hERG gating currents recorded from tsa201 cells were observed to activate on a millisecond time scale with time constants of on-gating current movement at 0 mV of 3.7 and 11.3 ms, much faster than ionic current activation, with bimodal voltage dependence (V 1/2-1 = –36.6 mV, k 1 = 8.3 mV, V 1/2-2 = 42.8 mV, k 2 = 15.5 mV, r² = 0.9912; A1=26%). Direct outward movement of the S4 domain as tracked by the modification of hERG channel I521C by MTSET was complete at 50 ms for pulses to 0 mV and within 25 ms for pulses to +60 mV. Conclusions This study represents the first report of hERG gating currents observed from mammalian cells and the first report of rapid voltage sensor modification associated with activation in hERG channels. Gating current recordings from tsa201 cells and the rate of MTSET modification of hERG voltage sensors suggest that the delayed activation kinetics of hERG cannot be attributed to slow voltage sensor movement but may be rate limited by a slow coupling process to the opening pore.

Identification and Characterization of a Compound That Protects Cardiac Tissue from Human Ether-à-go-go-related Gene (hERG)-related Drug-induced Arrhythmias

The human Ether-à-go-go-related gene (hERG)-encoded K(+) current, I(Kr) is essential for cardiac repolarization but is also a source of cardiotoxicity because unintended hERG inhibition by diverse pharmaceuticals can cause arrhythmias and sudden cardiac death. We hypothesized that a small molecule that diminishes I(Kr) block by a known hERG antagonist would constitute a first step toward preventing hERG-related arrhythmias and facilitating drug discovery. Using a high-throughput assay, we screened a library of compounds for agents that increase the IC(70) of dofetilide, a well characterized hERG blocker. One compound, VU0405601, with the desired activity was further characterized. In isolated, Langendorff-perfused rabbit hearts, optical mapping revealed that dofetilide-induced arrhythmias were reduced after pretreatment with VU0405601. Patch clamp analysis in stable hERG-HEK cells showed effects on current amplitude, inactivation, and deactivation. VU0405601 increased the IC(50) of dofetilide from 38.7 to 76.3 nM. VU0405601 mitigates the effects of hERG blockers from the extracellular aspect primarily by reducing inactivation, whereas most clinically relevant hERG inhibitors act at an inner pore site. Structure-activity relationships surrounding VU0405601 identified a 3-pyridiyl and a naphthyridine ring system as key structural components important for preventing hERG inhibition by multiple inhibitors. These findings indicate that small molecules can be designed to reduce the sensitivity of hERG to inhibitors.

N-Terminal Region Regulation of hERG Potassium Channel Gating: Contributions of the Eag/Pas Domain and the Proximal N-Terminus

The Human Ether-á-go-go Related Gene (hERG) encodes a voltage-activated K+ channel. hERG contributes to the repolarization of the ventricular action potential as part of the IKr current and has also been shown to modulate neuronal firing frequency. hERG gating is characterized by rapid inactivation upon depolarization and rapid recovery from inactivation and slow closing (deactivation) upon repolarization. These factors combine to create a resurgent hERG current, where the current amplitude is paradoxically larger with repolarization than with depolarization. Previous data have shown that the N-terminal region of hERG regulates gating kinetics, however the molecular mechanisms of this regulation are not fully understood. Deletion of the hERG N-terminus (amino acids 2-354) has been shown to speed channel deactivation and recovery from inactivation compared to that of wild-type hERG channels. Relative outward current amplitude is also increased during the depolarization phase, leading to reduced current rectification. A genetically encoded eag domain fragment (amino acids 1-135) was shown to restore slow deactivation to N-truncated channels by forming a stable interaction with the C-terminal cyclic nucleotide binding domain (CNBD). Our present study sought to further investigate the contributions of the eag domain and proximal N-terminal region (amino acids 136-354) to hERG gating kinetics. We coexpressed genetically encoded N-terminal region fragments with hERG channels bearing a deletion of the full N-terminus (D2-354) or the eag domain (D2-135) in Xenopus oocytes and measured current with two-electrode voltage-clamp recordings. Here we report that eag domain-containing fragments (N1-135, N1-354) were demonstrated to reduce relative outward current, slow deactivation, and slow recovery from inactivation, resulting in channels with properties similar to those measured in wild-type hERG. The proximal N-terminal region was shown to be involved in steady-state activation.

Bimodal Regulation of hERG Gating by the N-Terminal Tail Revealed by Voltage Clamp Fluorometry

The cytoplasmic N-terminal domain of human-ether-a-go-go related gene (hERG) K+ channels contains an amphipathic α-helix and an unstructured tail. Deletion of residues 2-9 (Δ2-9, the unstructured tail) or 2-25 (Δ2-25, α-helix and unstructured tail) both accelerated deactivation and slowed the rate of activation. However, while Δ2-9 shifted the V0.5 of activation in the depolarized direction, Δ2-25 did not, suggesting differential effects of the two deletions on the activation process.In this study we have used voltage clamp fluorometry to investigate the mechanism of how these N-terminal deletions affect voltage sensor movement during the activation process. The V0.5 of the fluorescence-voltage relationship for Δ2-9 was shifted to more depolarized potentials (18.2 ± 1.5 mV, n= 9) compared to control (0 ± 1.8 mV, n=12, ANOVA) indicating that Δ2-9 affects activation gating by shifting the voltage range over which the voltage sensor moves, while the V0.5 of activation for Δ2-25 (1 ± 2.9 mV, n=7) was not significantly different from control. In contrast, both Δ2-9 and Δ2-25 have slow rates of current activation with no observable effect on the fluorescent report of the rate of voltage sensor movement, suggesting that both Δ2-9 and Δ2-25 slowed rates of activation independent of the voltage sensor.Our data therefore suggests a bimodal regulation of activation gating by the N-terminal domain of hERG channels. We propose that the α-helical region modulates activation gating via an effect on movement of the voltage sensor while the N-terminal tail (residues 1-9) interacts with the cytoplasmic activation gate to regulate the rates of pore opening and closing.

Development and Validation of a high-throughput Structure-Activity Relationship Assay for the hERG Potassium Channel

Drug-induced prolongation of the QT interval via hERG block is a major cause of attrition in drug development. The advent of automated electrophysiology systems has greatly reduced this risk by enabling the detection of hERG block earlier in drug discovery. In this study, we have evaluated the suitability of the IonWorks Barracuda for the characterization of hERG biophysics and pharmacology. The IonWorks Barracuda is a second generation automated patch clamp system with increased throughput and capabilities, allowing 384 parallel recordings and up to 8 additions per recording. All experiments were conducted with CHO cells stably expressing hERG. Recordings were made in population patch clamp mode, where each current is measured from an average of up to 64 cells. We first examined hERG gating characteristics. HERG V1/2 for activation was −7.8 ± 1.8 mV (n=183) on the IonWorks Barracuda versus −17.5 ± 5.3 mV (n=9) by manual patch clamp. For pharmacological experiments, all drug additions were made in a cumulative fashion such that up to 384 dose-responses could be obtained in a single experiment. Seal resistances and currents were stable (<20% change) with up to 6 additions and 1-minute incubations per addition. Over 27 experiments, 96% of the recordings yielded full dose-response curves. HERG pharmacology was examined with a set of 353 compounds that included well-characterized hERG blockers. Astemizole, terfenadine and quinidine inhibited hERG currents with IC50 values of 159 nM, 224 nM and 2 microM, respectively (n=51, 10 & 18). This set of compounds was also tested with the PatchXpress automated electrophysiology system. We determined through statistical methods that the two automated systems provided equivalent results. In conclusion, this study showed that hERG biophysical and pharmacological characterizations can be conducted very efficiently with the IonWorks Barracuda.

Voltage-Dependent Gating of hERG Potassium Channels

The mechanisms by which voltage-gated channels sense changes in membrane voltage and energetically couple this with opening of the ion conducting pore has been the source of significant interest. In voltage-gated potassium (Kv) channels, much of our knowledge in this area comes from Shaker-type channels, for which voltage-dependent gating is quite rapid. In these channels, activation and deactivation are associated with rapid reconfiguration of the voltage-sensing domain unit that is electromechanically coupled, via the S4–S5 linker helix, to the rate-limiting opening of an intracellular pore gate. However, fast voltage-dependent gating kinetics are not typical of all Kv channels, such as Kv11.1 (human ether-à-go-go related gene, hERG), which activates and deactivates very slowly. Compared to Shaker channels, our understanding of the mechanisms underlying slow hERG gating is much poorer. Here, we present a comparative review of the structure–function relationships underlying activation and deactivation gating in Shaker and hERG channels, with a focus on the roles of the voltage-sensing domain and the S4–S5 linker that couples voltage sensor movements to the pore. Measurements of gating current kinetics and fluorimetric analysis of voltage sensor movement are consistent with models suggesting that the hERG activation pathway contains a voltage independent step, which limits voltage sensor transitions. Constraints upon hERG voltage sensor movement may result from loose packing of the S4 helices and additional intra-voltage sensor counter-charge interactions. More recent data suggest that key amino acid differences in the hERG voltage-sensing unit and S4–S5 linker, relative to fast activating Shaker-type Kv channels, may also contribute to the increased stability of the resting state of the voltage sensor.