Comparative effects of azimilide and ambasilide on the human ether-a-go-go-related gene (HERG) potassium channel.

OBJECTIVE: To evaluate the effects of azimilide and ambasilide on the biophysical properties of the human-ether-a-go-go-related (HERG) channel. METHODS: HERG was stably transfected into Chinese hamster ovary (CHO-K1) cells and currents were measured using a whole cell, voltage-clamp technique. RESULTS: Azimilide had a 'dual effect', inhibiting current at voltage steps above -40 mV and augmenting current at -40 and -50 mV. Tail current inhibition following a step to +30 mV did not vary with temperature (IC(50) 610 nM at 22 degrees C and 560 nM at 37 degrees C). The agonist effect at -50 mV was concentration-dependent and correlated with a hyperpolarizing shift in the V(1/2) of activation (r=0.98, P<0.05). Time constants of inactivation were faster and there was a -10 mV shift in the V(1/2) of steady state inactivation suggestive of open and inactivated state binding. By comparison, ambasilide inhibited HERG channels with lower potency (IC(50) 3.6 microM), in a voltage- and time-dependent but frequency-independent manner (0.03-1 Hz). Ambasilide had no effect on activation or inactivation gating but prolonged both fast and slow components of deactivation consistent with unbinding from the open state. The net effect of both drugs was similar during a voltage ramp which simulated a cardiac action potential. CONCLUSIONS: Inhibition of HERG channels by azimilide and ambasilide exhibits a similar time and voltage-dependence. While both exhibit affinity for the open state, azimilide also binds to inactivated channels.

Inhibition of HERG potassium channels by the antimalarial agent halofantrine.

Halofantrine is a widely used antimalarial agent which has been associated with prolongation of the 'QT interval' of the electrocardiogram (ECG), torsades de pointes and sudden death. Whilst QT prolongation is consistent with halofantrine-induced increases in cardiac ventricular action potential duration, the cellular mechanism for these observations has not been previously reported. The delayed rectifier potassium channel, I(Kr), is a primary site of action of drugs causing QT prolongation and is encoded by the human-ether-a-go-go-related gene (HERG). We examined the effects of halofantrine on HERG potassium channels stably expressed in Chinese hamster ovary (CHO-K1) cells. Halofantrine blocked HERG tail currents elicited on repolarization to -60 mV from +30 mV with an IC(50) of 196.9 nM. The therapeutic plasma concentration range for halofantrine is 1.67-2.98 microM. Channel inhibition by halofantrine exhibited time-, voltage- and use-dependence. Halofantrine did not alter the time course of channel activation or deactivation, but inactivation was accelerated and there was a 20 mV hyperpolarizing shift in the mid-activation potential of steady-state inactivation. Block was enhanced by pulses that render channels inactivated, and channel blockade increased with increasing duration of depolarizing pulses. We conclude that HERG channel inhibition by halofantrine is the likely underlying cellular mechanism for QT prolongation. Our data suggest preferential binding of halofantrine to the open and inactivated channel states.

Inhibition of the human ether-a-go-go-related gene (HERG) potassium channel by cisapride: affinity for open and inactivated states.

1 Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high affinity blockade of IKr (encoded by HERG). 2 In a chronic transfection model using CHO-K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC50 16. 4 nM at 20-22 degrees C and 23.6 nM at 37 degrees C). 3 Channel inhibition exhibited time-, voltage- and frequency-dependence. In an envelope of tails test, channel blockade increased from 27+/-8% after a 120 ms depolarizing step to 50+/-4% after a 1.0 s step. These findings suggested affinity for open and/or inactivated channel states. 4 Inactivation was significantly accelerated by cisapride in a concentration-dependent manner and there was a small (-7 mV) shift in the voltage dependence of steady state inactivation. 5 Channel blockade by cisapride was modulated by [K+]o, with a 26% reduction in the potency of channel blockade when [K+]o was increased from 1 to 10 mM. 6 In conclusion, HERG channel inhibition by cisapride exhibits features consistent with open and inactivated state binding and is sensitive to external potassium concentration. These features may have significant clinical implications with regard to the mechanism and treatment of cisapride-induced proarrhythmia.

Blockade by N-3 polyunsaturated fatty acid of the Kv4.3 current stably expressed in Chinese hamster ovary cells.

1. The Kv4.3 gene is believed to encode a large proportion of the transient outward current (Ito), responsible for the early phase of repolarization of the human cardiac action potential. There is evidence that this current is involved in the dispersion of refractoriness which develops during myocardial ischaemia and which predisposes to the development of potentially fatal ventricular tachyarrhythmias. 2. Epidemiological, clinical, animal, and cellular studies indicate that these arrhythmias may be ameliorated in myocardial ischaemia by n-3 polyunsaturated fatty acids (n-3 PUFA) present in fish oils. 3. We describe stable transfection of the Kv4.3 gene into a mammalian cell line (Chinese hamster ovary cells), and using patch clamp techniques have shown that the resulting current closely resembles human Ito. 4. The current is rapidly activating and inactivating, with both processes being well fit by double exponential functions (time constants of 3.8 +/- 0.2 and 5.3 +/- 0.4 ms for activation and 20.0 +/- 1.2 and 96.6+/-6.7 ms for inactivation at +45 mV at 23 degrees C). Activation and steady state inactivation both show voltage dependence (V1/2 of activation= -6.7+/-2.5 mV, V1,2 of steady state inactivation= -51.3+/-0.2 mV at 23 degrees C). Current inactivation and recovery from inactivation are faster at physiologic temperature (37 degrees C) compared to room temperature (23 degrees C). 5. The n-3 PUFA docosahexaenoic acid blocks the Kv4.3 current with an IC50 of 3.6 micromol L(-1). Blockade of the transient outward current may be an important mechanism by which n-3 PUFA provide protection against the development of ventricular fibrillation during myocardial ischaemia.

Inhibition of HERG channels stably expressed in a mammalian cell line by the antianginal agent perhexiline maleate.

Perhexiline has been used as an anti-anginal agent for over 25 years, and is known to cause QT prolongation and torsades de pointes. We hypothesized that the cellular basis for these effects was blockade of I(Kr). A stable transfection of HERG into a CHO-K1 cell line produced a delayed rectifier, potassium channel with similar properties to those reported for transient expression in Xenopus oocytes. Perhexiline caused voltage- and frequency-dependent block of HERG (IC50 7.8 microM). The rate of inactivation was increased and there was a 10 mV hyperpolarizing shift in the voltage-dependence of steady-state inactivation, suggestive of binding to the inactivated state. In conclusion, perhexiline potently inhibits transfected HERG channels and this is the probable mechanism for QT prolongation and torsades de pointes. Channel blockade shows greatest affinity for the inactivated state.

Differential effects of antiarrhythmic agents on post-pause repolarization in cardiac Purkinje fibres.

1. Marked action potential duration (APD) prolongation with agents such quinidine is often a precursor of early after-depolarizations and triggered activity, thought to be underlying mechanism of torsade de pointes. Episodes of torsade de pointes commonly occur following a pause. 2. We recently demonstrated that quinidine, but not disopyramide, produced marked further prolongation of APD immediately following pauses of 2-10s interpolated into a basic drive train in canine Purkinje fibres. 3. We report here experiments aimed at further elucidating the mechanisms of this phenomenon. 4. We used standard microelectrode techniques to record action potentials from canine Purkinje fibres driven at a baseline interstimulus interval (ISI) of 1000 ms. 5. We were able to reproduce the phenomenon of post-pause prolongation of APD with amitriptyline, which blocks both sodium and potassium channels, as does quinidine. Furthermore, we showed that the kinetics of interaction of amitriptyline, with the sodium channel, are similar to those known to exist for quinidine (time constant of recovery from blockade 2.3 +/- 0.6s). 6. In contrast, we were unable to reproduce post-pause prolongation of APD with three pure class III antiarrhythmic agents, D-sotalol, clofilium and dofetilide. 7. We propose that quinidine and amitriptyline behave similarly, in that they both produce two separate, opposing effects on APD. During a pause, the sodium channel-blocking action of these compounds diminishes exponentially, allowing the potassium channel blocking effect to become manifest as post-pause prolongation of APD. None of D-sotalol, clofilium or dofetilide exhibits significant sodium channel blockade and, thus, these agents do not manifest post-pause prolongation of repolarization. Disopyramide does produce sodium channel blockade, but recovery from this effect is much slower than for quinidine or amitriptyline (time constant 12-50s). Thus, we propose insufficient recovery occurs during the intervals under study to uncover the action potential-prolonging effect of the unopposed potassium channel blockade for disopyramide.

Effect of the class III antiarrhythmic agent E-4031 on the ATP-sensitive potassium channel in rabbit ventricular myocytes.

The class III antiarrhythmic drug E-4031, a known blocker of the delayed rectifier potassium channel (IK), might also be capable of blocking the ATP-sensitive potassium channel (IKATP). We examined this possibility by studying the effect of E-4031 on single IKATP channels in membrane patches excised from ventricular myocytes that were obtained by standard enzymatic dissociation techniques from New Zealand white rabbits. In inside-out patches, E-4031 caused a dose-dependent block of IKATP with an EC50 of 31 +/- 1 microM, Hill coefficient of 0.89 +/- 0.24 and no effect on channel conductance. Open dwell-time kinetics were fitted by two exponential components, with E-4031 causing reduction of the longer time constant. In outside-out patches, the concentration of E-4031 required to produce blockade was much higher. We conclude that E-4031 blocks the ATP-sensitive potassium channel and that it does so from within the cytoplasm, with one-to-one channel binding stoichiometry. Single channel conductance is unchanged, but the longer time constant for the open state is reduced, which suggests that E-4031 may be an open channel blocker of intermediate to slow time course.

Inhibition of ATP-sensitive potassium channels in cardiac myocytes by the novel class III antiarrhythmic agent MS-551.

The novel class III antiarrhythmic agent, MS-551, has recently been shown to attenuate the decrease in ventricular effective refractory period and to prevent the subsequent ventricular fibrillation induced by pinacidil and hypoxia in isolated perfused rabbit hearts (Friedrichs et al. 1994). We studied the effects of MS-551 on single ATP-sensitive potassium channels in isolated rabbit ventricular myocytes using standard patch-clamp methods. MS-551 in the range from 1 microM to 100 microM produced a concentration-dependent reduction of the open probability of the ATP-sensitive potassium channel, with an apparent ED50 of 30 microM. This reduced channel activity was due to a smaller number of channel openings per unit time, and the average duration of each opening of the channel was unaffected. This property of MS-551 is likely to be of most significance in ischaemic tissue, where the ATP-sensitive channels are thought to carry the predominant current that shortens the duration of the action potential.

Quinidine but not disopyramide prolongs cardiac Purkinje fiber action potentials after a pause.

Prolongation of the cardiac action potential (AP), leading eventually to early afterdepolarizations (EADs), is believed to underlie drug-induced long QT syndromes and torsade de pointes. Episodes of torsade de pointes frequently occur after a prolonged pause. We studied the effects of quinidine and disopyramide on AP duration (APD) in canine cardiac Purkinje fibers after pauses of 2,000-10,000 ms. Standard intracellular microelectrode techniques were used to record APs from canine Purkinje fibers at an interstimulus interval (ISI) of 1,000 ms. Pauses of 2,000-10,000 ms were introduced into the basic drive cycle in the presence and absence of subtherapeutic and therapeutic concentrations of quinidine and disopyramide. We observed a biphasic response in APD to quinidine and disopyramide at ISI = 1,000 ms. Quinidine but not disopyramide produced a marked dose- and time-dependent additional prolongation of APD immediately after the pauses. This effect was highly statistically significant. We conclude that disopyramide and quinidine have qualitatively different effects on APD after a pause and that this observation may cast some light on the apparently greater frequency of torsade de pointes occurring with quinidine than with disopyramide. Possible mechanisms include differential drug effects on outward potassium or inward sodium channels.

Effects of disopyramide and flecainide on the kinetics of inward rectifier potassium channels in rabbit heart muscle.

1. Standard patch-clamp techniques were used to study the interaction of therapeutic concentrations of flecainide and disopyramide with single inwardly-rectifying potassium channels in cell-attached membrane patches from rabbit ventricular myocytes. 2. Under drug-free conditions, the potassium channels had a conductance of 31 +/- 2 pS (n = 13), a mean open time of 230 +/- 6 ms (n = 11) recorded at the resting cell potential, and an open probability of 0.66 +/- 0.20 (n = 39). The resting potential of the cells studied was -68.5 +/- 3.6 mV (n = 32). 3. Disopyramide did not reduce the open probability of the channel when the cell was voltage-clamped at the resting cell potential. However, disopyramide increased the mean open time of the channel, recorded at the resting cell potential, by 15% at 5 microM and by 29% at 20 microM. The action potential prolonging actions of disopyramide in therapeutic concentrations appear not to be due to blocking the inward rectifier K+ channel. 4. Flecainide (3.0 microM, but not at 0.5 microM) decreased the open probability without changing the conductance of the channel, at 3 microM (51.0 +/- 7.2%, n = 6, P = 0.03) at the resting cell potential. Flecainide increased the mean open time of the channel, recorded at the resting cell potential, by 12% at 3.0 microM. 5. We propose that flecainide stabilized the inward rectifier K+ channel in an inactivated state, without plugging the conducting pore. In addition, it appeared to bind to an open conformation of the channel,since some of the reduction in open probability could be accounted for by the lengthening of the mean open time. The changes in open-state kinetics suggest that this binding may be in the region of the activation gate.

Action potential prolongation exhibits simple dose-dependence for sotalol, but reverse dose-dependence for quinidine and disopyramide: implications for proarrhythmia due to triggered activity.

Plasma drug concentrations in patients who develop torsade de pointes while receiving quinidine or disopyramide treatment have been reported to be usually in or below the therapeutic range, whereas patients developing the same complication during sotalol treatment usually have drug concentrations well above the therapeutic range. We wished to provide a cellular electrophysiologic rationale for this observation. Standard intracellular microelectrode techniques were used to record action potentials (APs) from canine Purkinje fibers at interstimulus intervals (ISI) of 1,000-6,000 ms, with and without three varying concentrations of quinidine, disopyramide, and sotalol. Cesium chloride 0.5 mM was added to reduce spontaneous diastolic depolarization. We observed a biphasic response in action potential duration (APD) to quinidine and disopyramide. Low concentrations tended to prolong APD, particularly at slower drive rates, whereas this effect tended to reverse as the concentration was increased. In contrast, sotalol produced a consistent, monophasic dose-dependent increase in APD across the therapeutic concentration range and well beyond it. We also observed an apparent increased likelihood of early afterdepolarizations (EADs), with or without triggered activity, at low concentrations of quinidine and disopyramide, with a trend toward reversal as the concentration was increased. We conclude that the biphasic dose response observed for APD with quinidine and disopyramide is due to the opposing effects of these agents on outward potassium and inward sodium currents and may cast some light on the clinical observation noted above. Sotalol on the other hand appears to produce EADs and triggered activity only at high concentrations.

Effects of hyperkalaemia on the depression of maximum rate of depolarization by class I antiarrhythmic agents in guinea-pig myocardium.

1 Standard microelectrode methods were used to record intracellular action potentials from strips of guinea-pig right ventricular myocardium superfused with either standard physiological saline ([K+] = 5.6 mM) or the same solution modified to contain [K+] = 11.2 mM. 2 The effects on action potential parameters of three therapeutic concentrations of mexiletine, quinidine and disopyramide were studied under both conditions at four different drive rates (interstimulus intervals = 2400, 1200, 600 and 300 ms). 3 Hyperkalaemia in the absence of drugs produced reductions in resting potential (-86.7 +/- 2.5 mV to -71.8 +/- 3.7 mV; n = 30; P < 0.001), maximum rate of depolarization (300 +/- 46.5 V s-1 to 205.6 +/- 37.6 V s-1; P < 0.0001), and action potential duration (205 +/- 26 ms to 188 +/- 32 ms; P < 0.05). 4 All three drugs produced increased depression of maximum rate of depolarization in hyperkalaemia compared to control conditions, but at all three concentrations this enhancement of effect was greater for mexiletine than for quinidine, with disopyramide exhibiting intermediate behaviour. 5 Mexiletine behaved very similarly to therapeutic concentrations of lignocaine as described in previous reports from this laboratory. 6 Quinidine behaved very similarly to Class Ic agents. 7 It is concluded that mexiletine demonstrated significantly greater selectivity for depolarized myocardium than quinidine and that this may have implications in terms of proarrhythmic potential. 8 Disopyramide exhibited intermediate selectivity for depolarized myocardium between mexiletine and quinidine.

Lidocaine shows greater selective depression of depolarized and acidotic myocardium than propafenone: possible implications for proarrhythmia.

We have previously shown reduced selectivity for depolarized and acidotic myocardium for encainide and flecainide compared to lidocaine and amiodarone. The present study aims to compare propafenone and two of its metabolites (5-OH-propafenone and N-despropyl-propafenone) to lidocaine in the same model. Standard microelectrode methods were used to record intracellular action potentials from strips of guinea pig right ventricular myocardium superfused with either standard physiological saline (pH 7.3; pO2 > 600 mm Hg; [K+] = 5.6 mM), or the same solution modified to produce either hyperkalemia (K+ = 11.2 mM), acidosis (pH = 6.3), or hypoxia (pO2 = 60 mm Hg). The effects on action potential parameters of three "therapeutic" concentrations of lidocaine, propafenone, and two of its metabolites were studied under all four conditions at four different drive rates from 200 to 25 beats/min. Hyperkalemia, in the absence of drugs, produced reductions in the resting potential (-86.7 +/- 2.5 to -71.8 +/- 3.7 mV) and the maximum rate of depolarization (Vmax, 300.0 +/- 46.5 to 205.6 +/- 37.6 V/s). All four drugs produced increased depression of Vmax in hyperkalemia and acidosis compared to control conditions, but it was a consistent finding that at concentrations that were approximately equipotent in control conditions, lidocaine produced greater increments in depression of Vmax in hyperkalemic and acidotic superfusate than did propafenone or either of its metabolites. Qualitatively similar results were obtained for both metabolites compared to lidocaine. Hypoxia produced no significant modulation of drug effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential effects on action potential duration of class IA, B and C antiarrhythmic drugs: modulation by stimulation rate and extracellular K+ concentration.

1. Standard microelectrode techniques were used to study the effects on the action potential duration (APD) of canine Purkinje fibres of a therapeutic concentration of nine Class I antiarrhythmic drugs. At an extracellular K+ concentration of 5.6 mmol/L all nine agents reduced APD at all drive rates studied (range of interstimulus intervals = 200-1000 ms). At lower levels of K+, quinidine (5 mumol/L) and disopyramide (10 mumol/L) (Class Ia agents) revealed dual effects on APD. At the lowest levels of K+ (2 mmol/L) and the longest interstimulus interval used (2000 ms), both agents significantly prolonged APD. Under all other conditions, APD was either unchanged or reduced. Lignocaine, 15 mumol/L (Class Ib agent) reduced APD at all rates and all K+ concentrations and this effect was greatest at the slowest rates. 2. Flecainide (1 mumol/L) (Class Ic) shortened APD at K+ = 5.6 and 4 mmol/L but had no effect at K+ = 2 mmol/L. 3. We conclude that these data result from opposing drug actions on inward sodium and outward potassium currents flowing during the plateau of the action potential. 4. Class Ia drugs exhibit significant depression of both currents, with the resultant effect on APD being modulated by external K+ concentration and drive rate. 5. Class Ib agents predominantly depress the sodium current and hence shorten APD, and Ic compounds have intermediate actions. 6. These differential effects on APD must be considered when planning antiarrhythmic therapy, and are directly relevant to the proarrhythmic propensities of these agents.

Effects of hyperkalemia, acidosis, and hypoxia on the depression of maximum rate of depolarization by class I antiarrhythmic drugs in guinea pig myocardium: differential actions of class Ib and Ic agents.

Standard microelectrode methods were used to record intracellular action potentials from strips of guinea pig right ventricular myocardium superfused with either standard physiological saline (pH 7.3; PO2 greater than 650 mm Hg; [K+] = 5.6 mM) or the same solution modified to produce either hyperkalemia ([ K+] = 11.2 mM), acidosis (pH = 6.3), or hypoxia (PO2 = 60 mm Hg). The effects on action potential parameters of three therapeutic concentration of lidocaine, flecainide, and encainide were studied under all four conditions at four different drive rates (interstimulus interval = 2,400, 1,200, 600, and 300 ms). Hyperkalemia in the absence of drugs produced reductions in resting potential (-87.9 +/- 3.8 to -74.6 +/- 3.3 mV), maximum rate of depolarization (316 +/- 68 to 240 +/- 12 V/s), and action potential duration (178 +/- 21 to 165 +/- 27 ms). All three drugs produced increased depression of Vmax in hyperkalemia compared to control conditions but, at all three concentrations and all four rates, this enhancement of effect was greater for lidocaine than for either of the other two agents (which did not differ significantly from each other; p less than 0.001). Similar though less marked effects were produced by acidosis (3.5 mV depolarization and 19% reduction in Vmax), and once again the depression of Vmax by lidocaine was enhanced more by this intervention than were the actions of encainide or flecainide (p less than 0.01). Hypoxia had no effect on action potential parameters other than duration and no significant modulation of drug actions was seen for this intervention.(ABSTRACT TRUNCATED AT 250 WORDS)