A benzopyran with antiarrhythmic activity is an inhibitor of Kir3.1-containing potassium channels.

Atrial fibrillation (AF) is the most commonly diagnosed cardiac arrhythmia and is associated with increased morbidity and mortality. Currently approved AF antiarrhythmic drugs have limited efficacy and/or carry the risk of ventricular proarrhythmia. The cardiac acetylcholine activated inwardly rectifying K+ current (IKACh), composed of Kir3.1/Kir3.4 heterotetrameric and Kir3.4 homotetrameric channel subunits, is one of the best validated atrial-specific ion channels. Previous research pointed to a series of benzopyran derivatives with potential for treatment of arrhythmias, but their mechanism of action was not defined. Here, we characterize one of these compounds termed Benzopyran-G1 (BP-G1) and report that it selectively inhibits the Kir3.1 (GIRK1 or G1) subunit of the KACh channel. Homology modeling, molecular docking, and molecular dynamics simulations predicted that BP-G1 inhibits the IKACh channel by blocking the central cavity pore. We identified the unique F137 residue of Kir3.1 as the critical determinant for the IKACh-selective response to BP-G1. The compound interacts with Kir3.1 residues E141 and D173 through hydrogen bonds that proved critical for its inhibitory activity. BP-G1 effectively blocked the IKACh channel response to carbachol in an in vivo rodent model and displayed good selectivity and pharmacokinetic properties. Thus, BP-G1 is a potent and selective small-molecule inhibitor targeting Kir3.1-containing channels and is a useful tool for investigating the role of Kir3.1 heteromeric channels in vivo. The mechanism reported here could provide the molecular basis for future discovery of novel, selective IKACh channel blockers to treat atrial fibrillation with minimal side effects.

Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting.

We report on wide-field time-correlated single photon counting (TCSPC)-based fluorescence lifetime imaging microscopy (FLIM) with lightsheet illumination. A pulsed diode laser is used for excitation, and a crossed delay line anode image intensifier, effectively a single-photon sensitive camera, is used to record the position and arrival time of the photons with picosecond time resolution, combining low illumination intensity of microwatts with wide-field data collection. We pair this detector with the lightsheet illumination technique, and apply it to 3D FLIM imaging of dye gradients in human cancer cell spheroids, and C. elegans.

Investigating dynamic protocol-dependence of hERG potassium channel inhibition at 37 degrees C: Cisapride versus dofetilide.

INTRODUCTION: Pharmacological inhibition of cardiac potassium channels encoded by hERG (human ether-à-go-go-related gene) is associated with QT interval prolongation and torsades de pointes arrhythmia. Electrophysiological assays of hERG channel inhibition are integral to the safety testing of novel drug candidates. This study was conducted to compare, for the high affinity hERG inhibitors dofetilide and cisapride, hERG blockade between action potential (AP) and conventional (step and step-ramp) screening waveforms. Furthermore, it evaluated dynamic (pulse-by-pulse) protocol-dependence of hERG channel inhibition by these drugs. METHODS: Whole-cell patch-clamp recordings were made at 37 degrees C from hERG-expressing HEK 293 cells. Half-maximal inhibitory concentrations (IC(50) values) for I(hERG) blockade were obtained using conventional voltage clamp and action potential clamp, using previously digitised ventricular and Purkinje fibre (PF) AP waveforms. RESULTS: A more marked variation in IC(50) values with different command waveforms was observed for cisapride (ranging from 7 to 72 nM) than for dofetilide (ranging from 4 to 15 nM), with higher IC(50)s obtained with AP than step or step-ramp commands. The two drugs differed little from one another in effects on voltage-dependent activation; however, I(hERG) blockade by each drug was initially voltage-dependent, but at steady-state was only voltage-dependent for cisapride. There was comparatively little difference between the two drugs in effects on I(hERG) availability or time constants of development of inactivation. Features of time-dependence of blockade and the use of protocols employing varying rest periods in drug or commands of alternating duration highlighted a pronounced ability of cisapride, but not dofetilide, to dissociate and reassociate from hERG on a pulse-by-pulse basis. DISCUSSION: Protocols described here that demonstrated dynamic variation (drug dissociation/reassociation) in hERG channel current blockade at 37 degrees C for cisapride may have future value for investigating drug interactions with the hERG channel. Downloadable digitised ventricular and PF AP waveforms that can be used in AP clamp experiments also accompany this article.

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656.

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.

Inhibition of the HERG K+ channel by the antifungal drug ketoconazole depends on channel gating and involves the S6 residue F656.

The mechanism of human ether-à-go-go-related gene (HERG) K+ channel blockade by the antifungal agent ketoconazole was investigated using patch-clamp recording from mammalian cell lines. Ketoconazole inhibited whole-cell HERG current (IHERG) with a clinically relevant half-maximal inhibitory drug concentration (IC50) value of 1.7 microM. The voltage- and time-dependent characteristics of IHERG blockade by ketoconazole indicated dependence of block on channel gating, ruling out a significant role for closed-state channel inhibition. The S6 HERG mutations Y652A and F656A produced approximately 4-fold and approximately 21-fold increases in IC50 for IHERG blockade, respectively. Thus, ketoconazole accesses the HERG channel pore-cavity on channel gating, and the S6 residue F656 is an important determinant of ketoconazole binding.

Clemastine, a conventional antihistamine, is a high potency inhibitor of the HERG K+ channel.

Human ether-à-go-go-related gene (HERG) encodes the alpha-subunit of channels carrying the cardiac rapid delayed K+ current (Ikr), which is a major determinant of the duration of ventricular action potentials (APs) and of the QT interval. This study investigated the effects on HERG channel current (IHERG) of clemastine, a "conventional" antihistamine that has been associated with delayed ventricular repolarization in vitro, but for which no adverse effects on the human QT interval have been reported. Whole-cell patch-clamp measurements of IHERG were made at 37 degrees C from human embryonic kidney (HEK 293) cells stably expressing HERG channels. IHERG tails at -40 mV following depolarizing pulses to +20 mV were inhibited by clemastine with an IC50 value of 12 nM; this drug concentration also produced a marked inhibition of peak IHERG elicited during an AP voltage-clamp command. Clemastine produced a reversible approximately -5 mV shift in the IHERG steady-state voltage-dependent activation curve, but voltage-dependence of inactivation was unaffected. Development of IHERG inhibition by clemastine showed strong time-dependence. The S6 point mutations Y652A and F656A greatly attenuated the inhibitory effect of clemastine. We conclude that clemastine is a high potency inhibitor of IHERG, that this action is contingent upon channel gating and that clemastine interacts with a high affinity drug-binding site in the HERG channel pore cavity. The disparity between clemastine's potent IHERG inhibition and a lack of QT-prolongation in normal clinical use underscores the need to interpret HERG IC50 data for novel compounds in the context of information from other safety assays.

The N588K-HERG K+ channel mutation in the 'short QT syndrome': mechanism of gain-in-function determined at 37 degrees C.

The idiopathic short QT syndrome (SQTS) is characterised by an abnormally short QT interval on the electrocardiogram and by an increased risk of arrhythmia and sudden death. One variant of the syndrome is linked to missense mutations that lead to a single amino-acid change (N588K; asparagine to lysine) in the S5-Pore linker region of the cardiac HERG K(+) channel. This study was performed in order to determine how the N588K mutation alters HERG channel current (I(HERG)) kinetics at mammalian physiological temperature. The whole-cell current-voltage (I-V) relation for wild-type (WT) I(HERG) measured from Chinese Hamster Ovary cells was maximal at approximately 0 mV and showed marked inward rectification positive to this. In contrast, N588K I(HERG) showed marked rectification only at +60 mV and at more positive voltages. The voltage dependence of activation of N588K-HERG did not differ significantly from that of WT-HERG. However, N588K I(HERG) had a significantly more positive inactivation V(0.5) (-8.14+/-0.82 mV) than did WT I(HERG) (-70.05+/-0.82 mV; P<0.001, unpaired t test; n=5 for each). Its P(Na)/P(K) ratio was also greater. The instantaneous I-V relation for N588K I(HERG) under action potential voltage clamp peaked at approximately +40 mV, compared to approximately -37 mV for WT-I(HERG). These findings underscore the importance of the S5-P linker in HERG channel function and indicate that N588K-HERG contributes increased repolarising current earlier in the ventricular action potential at physiological temperature due to a approximately +60 mV shift in voltage dependence of I(HERG) inactivation.

High affinity HERG K(+) channel blockade by the antiarrhythmic agent dronedarone: resistance to mutations of the S6 residues Y652 and F656.

Pharmacological inhibition of human-ether-a-go-go-related gene (HERG) K(+) channels by structurally and therapeutically diverse drugs is associated with the 'acquired' form of long QT syndrome and with potentially lethal cardiac arrhythmias. Two aromatic amino-acid residues (Y652 and F656) on the inner (S6) helices are considered to be key constituents of a high affinity drug binding site within the HERG channel pore cavity. Using wild-type (WT) and mutant HERG channels expressed in mammalian cell lines, we have investigated HERG channel current (I(HERG)) blockade at 37+/-1 degrees C by dronedarone (DRONED), a non-iodinated analogue of the Class III antiarrhythmic agent amiodarone (AMIOD). Under our conditions WT I(HERG) tails, measured at -40 mV following activating pulses to +30 mV, were blocked with IC(50) values of approximately 59 and 70 nM for DRONED and AMIOD, respectively. I(HERG) inhibition by DRONED was contingent upon channel gating, with block developing rapidly on membrane depolarization, but with no preference for activated over inactivated channels. High external [K(+)] (94 mM) reduced the potency of I(HERG) inhibition by both DRONED and AMIOD. Strikingly, mutagenesis to alanine of the S6 residue F656 (F656A) failed to eliminate blockade by both DRONED and AMIOD, whilst Y652A had comparatively little effect on DRONED but some effect on AMIOD. These findings demonstrate that high affinity drug blockade of I(HERG) can occur without a strong dependence on the Y652 and F656 aromatic amino-acid residues.

The low-potency, voltage-dependent HERG blocker propafenone--molecular determinants and drug trapping.

The molecular determinants of high-affinity human ether-a-go-go-related gene (HERG) potassium channel blockade by methanesulfonanilides include two aromatic residues (Phe656 and Tyr652) on the inner helices (S6) and residues on the pore helices that face into the inner cavity, but determinants for lower-affinity HERG blockers may be different. In this study, alanine-substituted HERG channel mutants of inner cavity residues were expressed in Xenopus laevis oocytes and were used to characterize the HERG channel binding site of the antiarrhythmic propafenone. Propafenone's blockade of HERG was strongly dependent on residue Phe656 but was insensitive or weakly sensitive to mutation of Tyr652, Thr623, Ser624, Val625, Gly648, or Val659 and did not require functional inactivation. Homology models of HERG based on KcsA and MthK crystal structures, representing the closed and open forms of the channel, respectively, suggest propafenone is trapped in the inner cavity and is unable to interact exclusively with Phe656 in the closed state (whereas exclusive interactions between propafenone and Phe656 are found in the open-channel model). These findings are supported by very slow recovery of wild-type HERG channels from block at -120 mV, but extremely rapid recovery of D540K channels that reopen at this potential. The experiments and modeling suggest that the open-state propafenone binding-site may be formed by the Phe656 residues alone. The binding site for propafenone (which may involve pi-stacking interactions with two or more Phe656 side-chains) is either perturbed or becomes less accessible because of closed-channel gating. This provides further evidence for the existence of gating-induced changes in the spatial location of Phe656 side chains.

Lidoflazine is a high affinity blocker of the HERG K(+)channel.

Lidoflazine is an antianginal calcium channel blocker that carries a significant risk of QT interval prolongation and ventricular arrhythmia. We investigated whether or not lidoflazine inhibits current through the rapid delayed rectifier K(+) channel alpha subunit (encoded by HERG - human ether-a-go-go-related gene), since this channel has been widely linked to drug-induced QT-prolongation. Lidoflazine inhibited potently HERG current (I(HERG)) recorded from HEK 293 cells stably expressing wild-type HERG (IC(50) of approximately 16 nM). It was approximately 13-fold more potent against HERG than was verapamil under similar conditions. On membrane depolarization, I(HERG) inhibition developed gradually, ruling out closed-channel state dependent inhibition. The effect of command voltage on the drug's action suggested that lidoflazine preferentially inhibits activated/open HERG channels. The S6 mutation Y652A largely eliminated the inhibitory action of lidoflazine, whilst the F656A mutation also reduced blocking potency. We conclude: first, that lidoflazine produces high affinity blockade of the alpha subunit of the HERG channel by binding to aromatic amino acid residues within the channel pore and, second, that this is likely to represent the molecular mechanism of QT interval prolongation by this drug.

Blockade of HERG potassium currents by fluvoxamine: incomplete attenuation by S6 mutations at F656 or Y652.

1. Pharmacological blockade of the Human ether-a-go-go related gene (HERG) potassium channel is commonly linked with acquired long QT syndrome and associated proarrhythmia. The objectives of this study were (i) to identify and characterise any inhibitory action on HERG of the selective-serotonin re-uptake inhibitor fluvoxamine, (ii) to then determine whether fluvoxamine shared the consensus molecular determinants of HERG blockade of those drugs so far tested. 2. Heterologous HERG potassium current (I(HERG)) was measured at 37 degrees C, using the whole-cell patch-clamp technique, from a mammalian cell line (Human embryonic kidney 293) expressing HERG channels. I(HERG) tails, following repolarisation from +20 to -40 mV, were blocked by fluvoxamine with an IC(50) of 3.8 micro M. 3. Blockade of wild-type HERG was of extremely rapid onset (within 10 ms) and showed voltage dependence, with fluvoxamine also inducing a leftward shift in voltage-dependent activation of I(HERG). Characteristics of block were consistent with a component of closed channel (or extremely rapidly developing open channel) blockade and dependence on open and inactivated channel states. The attenuated-inactivation mutation S631A partially reduced the blocking effect of fluvoxamine. 4. The S6 mutations, Y652A and F656A, and the pore helix mutant S631A only partially attenuated blockade by fluvoxamine at concentrations causing profound blockade of wild-type HERG. 5. All HERG-blocking pharmaceuticals studied to date have been shown to block F656 mutant channels with over 100-fold reduced potency compared to their blockade of the wild-type channel. Fluvoxamine is therefore quite distinct in this regard from previously studied agents.

Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin.

The scorpion toxin peptide BeKm-1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether-a-go-go-related gene potassium current (I(HERG)) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I(HERG) by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use- and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.

Characterisation of recombinant HERG K+ channel blockade by the Class Ia antiarrhythmic drug procainamide.

Class Ia antiarrhythmic drugs, including procainamide (PROC), are associated with cardiac sodium channel blockade, delayed ventricular repolarisation and with a risk of ventricular pro-arrhythmia. The HERG K(+) channel is frequently linked to drug-induced pro-arrhythmia. Therefore, in this study, interactions between PROC and HERG K(+) channels were investigated, with particular reference to potency and mechanism of drug action. Whole-cell patch-clamp recordings of HERG current (I(HERG)) were made at 37 degrees C from human embryonic kidney (HEK 293) cells stably expressing the HERG channel. Following activating pulses to +20 mV, I(HERG) tails were inhibited by PROC with an IC(50) value of approximately 139 microM. I(HERG) blockade was found to be both time- and voltage-dependent, demonstrating contingency upon HERG channel gating. However, I(HERG) inhibition by PROC was relieved by depolarisation to a highly positive membrane potential (+80 mV) that favoured HERG channel inactivation. These data suggest that PROC inhibits the HERG K(+) channel by a primarily 'open' or 'activated' channel state blocking mechanism and that avidity of drug-binding is decreased by extensive I(HERG) inactivation. The potency of I(HERG) blockade by PROC is much lower than for other Class Ia agents that have been studied previously under analogous conditions (quinidine and disopyramide), although the blocking mechanism appears similar. Thus, differences between the chemical structure of PROC and other Class Ia antiarrhythmic drugs may help provide insight into chemical determinants of blocking potency for agents that bind to open/activated HERG channels.

Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K+ channels expressed in mammalian cell lines and Xenopus oocytes.

The majority of drugs associated with QT interval prolongation share an ability to inhibit ionic currents passed by HERG potassium channels. One method of screening new chemical entities (NCEs) for QT prolonging potential is therefore to use heterologous systems expressing HERG channels. Such systems are also of value in the understanding of the function, kinetics, sorting, pharmacological sensitivities, and important molecular determinants of the HERG potassium channel. The methods for incorporating the HERG potassium channel into cells and measuring the consequent current are a mixture of techniques that are standard (for heterologous expression of most ion channels) and individualised to HERG. This review presents a selection of the most commonly used methods for examining heterologous HERG currents, as well as introducing some of the technical problems that may be encountered and their solutions. In mammalian cell lines, problems such as fragile membranes, high leak currents, inability to form a gigaseal, diminished HERG current, endogenous transient outward current, altered kinetics, and even occasional run down can interfere with measurements. In Xenopus oocytes, endogenous chloride currents, insufficient superfusate flow, diminished HERG current and HERG current 'run up' may create difficulties.