Electrophysiological characterization of the hERG R56Q LQTS variant and targeted rescue by the activator RPR260243

Human Ether-à-go-go (hERG) channels contribute to cardiac repolarization, and inherited variants or drug block are associated with long QT syndrome type 2 (LQTS2) and arrhythmia. Therefore, hERG activator compounds present a therapeutic opportunity for targeted treatment of LQTS. However, a limiting concern is over-activation of hERG resurgent current during the action potential and abbreviated repolarization. Activators that slow deactivation gating (type I), such as RPR260243, may enhance repolarizing hERG current during the refractory period, thus ameliorating arrhythmogenicity with reduced early repolarization risk. Here, we show that, at physiological temperature, RPR260243 enhances hERG channel repolarizing currents conducted in the refractory period in response to premature depolarizations. This occurs with little effect on the resurgent hERG current during the action potential. The effects of RPR260243 were particularly evident in LQTS2-associated R56Q mutant channels, whereby RPR260243 restored WT-like repolarizing drive in the early refractory period and diastolic interval, combating attenuated protective currents. In silico kinetic modeling of channel gating predicted little effect of the R56Q mutation on hERG current conducted during the action potential and a reduced repolarizing protection against afterdepolarizations in the refractory period and diastolic interval, particularly at higher pacing rates. These simulations predicted partial rescue from the arrhythmic effects of R56Q by RPR260243 without risk of early repolarization. Our findings demonstrate that the pathogenicity of some hERG variants may result from reduced repolarizing protection during the refractory period and diastolic interval with limited effect on action potential duration, and that the hERG channel activator RPR260243 may provide targeted antiarrhythmic potential in these cases.     

Calcium-activated conductance in skate electroreceptors: current clamp experiments

When current clamped, skate electroreceptor epithelium produces large action potentials in response to stimuli that depolarize the lumenal faces of the receptor cells. With increasing stimulus strength these action potentials become prolonged. When the peak voltage exceeds about 140 mV the repolarizing phase is blocked until the end of the stimulus. Perfusion experiments show that the rising phase of the action potential results from an increase in calcium permeability in the lumenal membranes. Perfusion of the lumen with cobalt or with a zero calcium solution containing EGTA blocks the action potential. Perfusion of the lumen with a solution containing 10 mM Ca and 20 mM EGTA initially slows the repolarizing process at all voltages and lowers the potential at which it is blocked. With prolonged perfusion, repolarization is blocked at all voltages. When excitability is abolished by perfusion with cobalt, or with a zero calcium solution containing EGTA, no delayed rectification occurs. We suggest that repolarization during the action potential depends on an influx of calcium into the cytoplasm, and that the rate of repolarization depends on the magnitude of the inward calcium current. Increasingly large stimuli reduce the rate of repolarization by reducing the driving force for calcium, and then block repolarization by causing the lumenal membrane potential to exceed ECa. Changes in extracellular calcium affect repolarization in a manner consistent with the resulting change in ECa.     

Different effects of blocked potassium channels on action potentials, accommodation, adaptation and anode break excitation in human motor and sensory myelinated nerve fibres: computer simulations

 Action potentials and electrotonic responses to 300-ms depolarizing and hyperpolarizing currents for human motor and sensory myelinated nerve fibres have been simulated on the basis of double cable models. The effects of blocked nodal or internodal potassium (fast or slow) channels on the fibre action potentials, early and late adaptations to 30-ms suprathreshold slowly increasing depolarizing stimuli have been examined. The effects of the same channels on accommodation after the termination of a prolonged (100 ms) hyperpolarizing current pulse have also been investigated. By removing the nodal fast potassium conductance the action potentials of the sensory fibres are considerably broader than those of the motor neurons. For both types of fibres, the blocked nodal slow potassium channels have a substantially smaller effect on the action potential repolarization. When the suprathreshold depolarizing current intensity is increased, the onset of the spike burst occurs sooner, which is common in the behaviour of the fibres. The most striking differences in the burst activity during early adaptation have been found between the fibres when the nodal fast potassium channels are blocked. The results obtained confirm the fact that the motor fibres adapt more quickly to sustained depolarizing current pulses than the sensory ones. The results also show that normal human motor and sensory fibres cannot be excited by a 100-ms hyperpolarizing current pulse, even at the threshold level. When removing the potassium channels in the nodal or internodal axolemma, the posthyperpolarization increase in excitability is small, which is common in the behaviour of the fibres. However, anode break excitation can be simulated in the fibres with simultaneous removal of the potassium channels under the myelin sheath, and this is more pronounced in the human sensory fibres than in motor fibres. This phenomenon can also be found when the internodal and some of the nodal (fast or slow) potassium channels are simultaneously blocked. Received: 8 November 1999 / Accepted in revised form: 29 February 2000     

Control of action potential duration by calcium ions in cardiac Purkinje fibers

It is well known that cardiac action potentials are shortened by increasing the external calcium concentration (Cao). The shortening is puzzling since Ca ions are thought to carry inward current during the plateau. We therefore studied the effects of Cao on action potentials and membrane currents in short Purkinje fiber preparations. Two factors favor the earlier repolarization. First, calcium-rich solutions generally raise the plateau voltage; in turn, the higher plateau level accelerates time- and voltage-dependent current changes which trigger repolarization. Increases in plateau height imposed by depolarizing current consistently produced shortening of the action potential. The second factor in the action of Ca ions involves iK1, the background K current (inward rectifier). Raising Cao enhances iK1 and thus favors faster repolarization. The Ca-sensitive current change was identified as an increase in iK1 by virtue of its dependence on membrane potential and Ko. A possible third factor was considered and ruled out: unlike epinephrine, calcium-rich solutions do not enhance slow outward plateau current, ikappa. These results are surprising in showing that calcium ions and epinephrine act quite differently on repolarizing currents, even though they share similar effects on the height and duration of the action potential.     

Simulations of propagated mouse ventricular action potentials: effects of molecular heterogeneity

The molecular heterogeneity of repolarizing currents produces significant spatial heterogeneity and/or dispersion of repolarization in many mammalian cardiac tissues. Transgenic mice are prominent experimental models for the study of the molecular basis of repolarization and arrhythmias. However, it is debated whether the small mouse heart can sustain physiologically relevant heterogeneity of repolarization. We used a comprehensive model of the mouse action potential (AP) to predict how small a region of the cardiac tissue can maintain spatial gradients of repolarization due to differential expression of channels. Our simulations of a one-dimensional multicellular ring or cable predict that substantial gradients in repolarization and intracellular Ca(2+) concentration transients can be maintained through heterogeneity of expression of K(+) channels in distances of approximately 10 cells that are sufficient to block propagation. The abruptness of expression gradients and the site of stimulation can cause Ca(2+) transient oscillations and affect the stability of Ca(2+) dynamics and AP propagation. Two different mechanisms of instability of AP propagation in one-dimensional cable occur at fast pacing rates. Transitions from periodic activity to alternans or to irregular behavior were observed. Abrupt gradients of channel expression can cause alternans at slower pacing rates than gradual changes. Our simulations demonstrate the importance of incorporating realistic Ca(2+) dynamics and current densities into models of propagated AP. They also emphasize that microscopic aspects of tissue organization are important for predicting large-scale propagation phenomena. Finally, our results predict that the mouse heart should be able to sustain substantial molecularly based heterogeneity of repolarization.     

Impact of Hypokalemia on Electromechanical Window,Excitation Wavelength and Repolarization Gradients in Guinea-Pig and Rabbit Hearts

Normal hearts exhibit a positive time difference between the end of ventricular contraction and the end of QT interval, which is referred to as the electromechanical (EM) window. Drug-induced prolongation of repolarization may lead to the negative EM window, which was proposed to be a novel proarrhythmic marker. This study examined whether abnormal changes in the EM window may account for arrhythmogenic effects produced by hypokalemia. Left ventricular pressure, electrocardiogram, and epicardial monophasic action potentials were recorded in perfused hearts from guinea-pig and rabbit. Hypokalemia (2.5 mM K⁺) was found to prolong repolarization, reduce the EM window, and promote tachyarrhythmia. Nevertheless, during both regular pacing and extrasystolic excitation, the increased QT interval invariably remained shorter than the duration of mechanical systole, thus yielding positive EM window values. Hypokalemia-induced arrhythmogenicity was associated with slowed ventricular conduction, and shortened effective refractory periods, which translated to a reduced excitation wavelength index. Hypokalemia also evoked non-uniform prolongation of action potential duration in distinct epicardial regions, which resulted in increased spatial variability in the repolarization time. These findings suggest that arrhythmogenic effects of hypokalemia are not accounted for by the negative EM window, and are rather attributed to abnormal changes in ventricular conduction times, refractoriness, excitation wavelength, and spatial repolarization gradients.     

Refractoriness of cardiac muscle as affected by intercalated disks: a model study implications for fibrillation and defibrillation

It is generally agreed that inhomogeneities of the recovery process in cardiac tissue play an important role in the genesis of reentrant arrhythmias. Regarding cardiac muscle as an assembly of discrete cells connected by gap junctions, differences in recovery may result from a nonuniformity of membrane or cable properties. In this study, a computer model of a one-dimensional cardiac muscle fiber including a periodic intercalated disk structure is used to study the influence of disk resistance (Rj) and stimulus strength (J) on refractoriness. Stimulating currents are applied externally in a bipolar arrangement. The basic effect of a current pulse is local de- and hyperpolarizations at the ends of an individual cell. Polarization develops very rapidly and increases with increasing values of Rj or J so that an interaction with membrane current kinetics becomes possible. When a premature stimulus is applied during repolarization of a conditioning action potential, multiple Na currents can occur, either caused by depolarization of the cathodal end of a cell or in the form of anode break excitation at the hyperpolarized end. Those currents affect the response of a fiber such that, at a given value of J, the refractory period is shortened by an increase in Rj. In a ring fiber model with different Rj values in the two halves of ring an extrastimulus timed between the refractory periods of the two branches results in a sustained circus movement. Varying stimulus strength yields an upper limit of vulnerability characterized by a "synchronized extrasystole". The ring model also implies the suppression of circus movement by an external shock. The minimal shock strength required for suppression is close to the upper limit of vulnerability. The simulations suggest that discrete effects of junctional resistance may be involved in fibrillation and defibrillation.     

Dynamics of early afterdepolarization-mediated triggered activity in cardiac monolayers

Early afterdepolarizations (EADs) are voltage oscillations that occur during the repolarizing phase of the cardiac action potential and cause cardiac arrhythmias in a variety of clinical settings. EADs occur in the setting of reduced repolarization reserve and increased inward-over-outward currents, which intuitively explains the repolarization delay but does not mechanistically explain the time-dependent voltage oscillations that are characteristic of EADs. In a recent theoretical study, we identified a dual Hopf-homoclinic bifurcation as a dynamical mechanism that causes voltage oscillations during EADs, depending on the amplitude and kinetics of the L-type Ca(2+) channel (LTCC) current relative to the repolarizing K(+) currents. Here we demonstrate this mechanism experimentally. We show that cardiac monolayers exposed to the LTCC agonists BayK8644 and isoproterenol produce EAD bursts that are suppressed by the LTCC blocker nitrendipine but not by the Na(+) current blocker tetrodoxin, depletion of intracellular Ca(2+) stores with thapsigargin and caffeine, or buffering of intracellular Ca(2+) with BAPTA-AM. These EAD bursts exhibited a key dynamical signature of the dual Hopf-homoclinic bifurcation mechanism, namely, a gradual slowing in the frequency of oscillations before burst termination. A detailed cardiac action potential model reproduced the experimental observations, and identified intracellular Na(+) accumulation as the likely mechanism for terminating EAD bursts. Our findings in cardiac monolayers provide direct support for the Hopf-homoclinic bifurcation mechanism of EAD-mediated triggered activity, and raise the possibility that this mechanism may also contribute to EAD formation in clinical settings such as long QT syndromes, heart failure, and increased sympathetic output.     

Elevated potassium shortens action potential duration by altering outward currents in chick dorsal root ganglia neurons

Dissociated embryonic chick dorsal root ganglion (DRG) neurons maintained in culture exhibit a mixed Na+/Ca2+ action potential. The characteristic "shoulder" on the repolarizing phase is due to the relatively prolonged inward Ca2+ current. DRG neurons grown in an elevated K+ medium (25 versus. 5 mM) lack the plateau phase of the action potential. Voltage-clamp analysis showed that this plastic change in action potential duration is not due to the loss of the inward Ca2+ current but is partly due to the appearance of a Ca2(+)-dependent, 4-aminopyridine-(4-AP)-sensitive transient outward current. Faster activation of the purely voltage-dependent delayed rectifier outward current also contributes to the rapid repolarization observed in neurons cultured in elevated K+ medium.     

Action potential refractory period in axonal demyelination: a computer simulation

Axonal demyelination leads to an increase in the refractory period for propagation of the action potential. Computer simulations were used to investigate the mechanism by which changes in the passive properties of the internodal membrane increase the refractory period. The properties of the voltage dependent ion channels can be altered to restore conduction in demyeliated nerve fibers. The ability of these alterations to decrease the refractory period of demyelinated model nerve fibers was compared. The model nerve fiber contained six nodes. The action potential was stimulated at node one and propagated to node six. The internode between nodes three and four was demyelinated in a graded manner. The absolute refractory period for propagation of the action potential through the demyelinated internode increased as the number of myelin wraps was reduced to less than 25% of the normal value. The increase in refractory period was found to be due to a reduction in the rate or repolarization of the action potential at node three. The delay in repolarization reduced the rate of recovery of inactivated Na channels and slowed the closing of K channels. The rate of repolarization of node three was reduced by the conduction delay for the depolarization of node four caused by demyelination of the preceeding internode. In these simulations the increase in refractory period due to demyelination was eliminated by slowing the onset of Na channel inactivation. A small reduction of the K conductance also decreased the refractory period. However, larger reductions eliminated this effect.     

Chloride Currents in Skeletal Muscles of Bufo arenarum

Cl⁻ currents (I _Cl) were measured in short fibers (1–2 mm) from the lumbricalis muscle of toads (Bufo arenarum) with two microelectrodes (15°C). Initially the fibers were equilibrated in a high K⁺-containing solution: (mm) K₂SO₄ 68; Na₂SO₄ 20; KCl 60; CaSO₄ 8; MgSO₄ 1; HEPES 2.5. Constant pulses were applied when all the external K⁺ was replaced by Cs⁺: Cs₂SO₄ 68; Na₂SO₄ 20; CsCl 60; CaSO₄ 8; HEPES 2.5 (pH 7.5). Under these conditions about 80–90% of the current is carried by Cl⁻. The current-voltage relation is almost linear implying constant conductance and hence voltage-independent permeability. The voltage dependence of the net Cl⁻ current could be fitted by constant field equation with a P _Cl of 3.3 × 10⁻⁶ cm/sec. In a separate group of experiments a two-pulse technique was used to estimate the availability and the inactivation of the initial I _Cl during a test pulse. After returning the potential to the holding potential for various times, test pulses of the same amplitude and duration of the prepulses were applied. The initial current during the test pulse was 70% of the initial current during the prepulse and the recovery was complete in less than 300 msec with a linear relationship between the current during the test pulse and the amplitude of the preceding prepulse. When the test pulses were preceded by a positive prepulse, the initial current for any given test pulse was larger than with a negative prepulse. If we assumed that the initial current during the test pulse is a measure of the number of channels open at the end of the prepulse, these results suggest that hyperpolarizing pulses inactivate and depolarizing prepulses activate the I _Cl. Received: 31 March 1995/Revised: 27 October 1995     

On reinforcement and interference between stimuli

It is shown that the current “two-factor” theory of nerve excitation can account for sustained inhibition or enhancement by a sequence of stimulus pulses, and for the decrease in the reinforcement period with each successive pulse of the train.     

Kv3.4 channel function and dysfunction in nociceptors

Recently, we reported the isolation of the Kv3.4 current in dorsal root ganglion (DRG) neurons and described dysregulation of this current in a spinal cord injury (SCI) model of chronic pain. These studies strongly suggest that rat Kv3.4 channels are major regulators of excitability in DRG neurons from pups and adult females, where they help determine action potential (AP) repolarization and spiking properties. Here, we characterized the Kv3.4 current in rat DRG neurons from adult males and show that it transfers 40–70% of the total repolarizing charge during the AP across all ages and sexes. Following SCI, we also found remodeling of the repolarizing currents during the AP. In the light of these studies, homomeric Kv3.4 channels expressed in DRG nociceptors are emerging novel targets that may help develop new approaches to treat neuropathic pain.     

New Aspects of HERG K+ Channel Function Depending upon Cardiac Spatial Heterogeneity

HERG K⁺ channel, the genetic counterpart of rapid delayed rectifier K⁺ current in cardiac cells, is responsible for many cases of inherited and drug-induced long QT syndromes. HERG has unusual biophysical properties distinct from those of other K⁺ channels. While the conventional pulse protocols in patch-clamp studies have helped us elucidate these properties, their limitations in assessing HERG function have also been progressively noticed. We employed AP-clamp techniques using physiological action potential waveforms recorded from various regions of canine heart to study HERG function in HEK293 cells and identified several novel aspects of HERG function. We showed that under AP-clamp I_HERG increased gradually with membrane repolarization, peaked at potentials around 20–30 mV more negative than revealed by pulse protocols and at action potential duration (APD) to 60%-70% full repolarization, and fell rapidly at the terminal phase of repolarization. We found that the rising phase of I_HERG was conferred by removal of inactivation and the decaying phase resulted from a fall in driving force, which were all determined by the rate of membrane repolarization. We identified regional heterogeneity and transmural gradient of I_HERG when quantified with the area covered by I_HERG trace. In addition, we observed regional and transmural differences of I_HERG in response to dofetilide blockade. Finally, we characterized the influence of HERG function by selective inhibition of other ion currents. Based on our results, we conclude that the distinct biophysical properties of HERG reported by AP-clamp confer its unique function in cardiac repolarization thereby in antiarrhythmia and arrhythmogenesis.     

Role of the inward K rectifier in the repetitive activity at the depolarized level in single ventricular myocytes

The role of the inward K⁺ rectifier in the repetitive activity at depolarized levels was studied in guinea pig single ventricular myocytes by voltage- and current-clamp methods. In action potentials arrested at the plateau by a depolarizing current, small superimposed hyperpolarizing currents caused much larger voltage displacements than at the resting potential and sometimes induced a regenerative repolarization. Around –20 mV, sub- and suprathreshold repetitive inward currents were found. In the same voltage range, small hyperpolarizing currents reversed their polarity. During depolarizing voltage-clamp ramps, around –20 mV there was a sudden decrease in the outward current (I_ns: current underlying the negative slope in the inward K⁺ rectifier steady state I–V relation). During repolarizing ramps, the reincrease in outward current was smaller and slower. During depolarizing and repolarizing current ramps, sudden voltage displacements showed a similar asymmetry. Repetitive I_ns could continue as long as the potential was kept at the level at which they appeared. Depolarizing voltage-clamp steps also caused repetitive I_ns and depolarizing current steps induced repetitive slow responses. Cadmium and verapamil reduced I_ns amplitude during the depolarizing ramp. BRL 34915 (cromakalim), an opener of the ATP-sensitive K⁺ channel, eliminated the negative slope and I_ns, whereas barium increased I_ns frequency (an effect abolished by adding BRL). Depolarization-induced slow responses persisted in an NaCl-Ca-free solution. Thus, the mechanism of repetitive activity at the depolarized level appears to be related to the presence of the negative slope in the inward K⁺ rectifier I–V relation.     

Phase resetting of embryonic chick atrial heart cell aggregates. Experiment and theory.

The influence of brief duration current pulses on the spontaneous electrical activity of embryonic chick atrial heart cell aggregates was investigated experimentally and theoretically. A pulse could either delay or advance the time of the action potential subsequent to the pulse depending upon the time in the control cycle at which it was applied. The perturbed cycle length throughout the transition from delay to advance was a continuous function of the time of the pulse for small pulse amplitudes, but was discontinuous for larger pulse amplitudes. Similar results were obtained using a model of the ionic currents which underlie spontaneous activity in these preparations. The primary ion current components which contribute to phase resetting are the fast inward sodium ion current, INa, and the primary, potassium ion repolarization current, IX1. The origin of the discontinuity in phase resetting of the model can be elucidated by a detailed examination of the current-voltage trajectories in the region of the phase response curve where the discontinuity occurs.     

Contractile activation phenomena in voltage-clamped barnacle muscle fiber

Tension development in voltage-clamped barnacle muscle fibers occurs with depolarizing pulses so small as not to activate the potassium and calcium conductance systems. Peak tension and the tension time integral appear to be graded by both amplitude and duration of the depolarizing pulses. Subthreshold depolarizing conditioning pulses shorter than 500 ms potentiate the response to a given test pulse. This effect diminishes and reverts when the duration of the conditioning pulse is increasingly prolonged. The relationship between fiber membrane potential and tension developed in response to depolarizing pulses is described by an S-shaped curve. The tension saturates at a membrane potential of about +10 mV (inside positive). For a given pulse duration the saturation value remains constant even when the fiber interior reaches a value of +230 mV, which is well above what may be estimated to be the equilibrium potential of calcium ions (Eca = +120). In the presence of 5 mM external procaine, the shape of the tension-potential curve changes; the maximum value tension besides being diminished is not sustained by falls when the potential approaches the estimated value for Eca. These results suggest that under physiological conditions the contractile activator is probably released from an internal store, and that the calcium entering the fiber as inward current does not play a direct major role in contractile activation.     

Computer simulation of acidosis-induced abnormal repolarization and repetitive activity in dog Purkinje fibers

Equations described by Mc Allister et al. (1975) were used to simulate the abnormal repolarization phase of the action potential (early after depolarization or hump), which often occurs in isolated dog Purkinje fibers submitted to acid media and can result in re-excitation. The development of humps was simulated by modifying the īK2-Em relationship in such a way that the resulting repolarizing current was increased at low membrane potential and decreased at higher (more negative) membrane potential. Beyond a certain size, humps triggered single, or multiple re-excitations. Hump size was very sensitive to small changes in īK2 and iK1, moderately sensitive to changes in īx1 and almost insensitive to changes in ix2. Negative shifts in the s infinity-Em relationship induced slowing of the basic frequency and only a small decrease in hump size. Decreasing gsi and gNa exerted inhibitory effects on hump development, the latter effect being larger than the former. The steady-state inward sodium current resulting from the overlap of m infinity-Em and h infinity-Em relationships appeared of particular importance in the development of humps.     

Demonstration of an “Excitation-Contraction Recoupling” Mechanism in Mammalian Ventricular Myocardium

A PROCESS called “excitation-contraction coupling” has been generally accepted to take place only in the direction of excitation to contraction. Through this mechanism a propagated action potential initiates an active state in skeletal or cardiac muscle and the muscle contracts. We propose that, in the mammalian ventricular myocardium at least, the process is not unidirectional and an important reverse coupling between the contractile system and the excitable plasma membrane has been overlooked. Through this feedback interaction the mode of contraction (that is, isotonic or isometric) not only determines the instantaneous electrical state of the plasma membrane, but also influences the mechanical events of the subsequent beats. Thus when Kaufmann et al.¹ recorded intracellular action potentials from cat papillary muscle, the time course of the repolarization was altered depending on the mode of contraction. Some kind of contraction-excitation feedback has also been suggested by Stauch² and Lab^3,4. They showed a difference in the shape of the monophasic action potential, as recorded by a suction electrode, when comparing isotonic and isovolumic contraction of the intact ventricle. But their experimental conditions did not allow satisfactory analysis of the phenomenon.     

Calcium-activated potassium conductance in presynaptic terminals at the crayfish neuromuscular junction

Membrane potential changes that typically evoke transmitter release were studied by recording intracellularly from the excitor axon near presynaptic terminals of the crayfish opener neuromuscular junction. Depolarization of the presynaptic terminal with intracellular current pulses activated a conductance that caused a decrease in depolarization during the constant current pulse. This conductance was identified as a calcium-activated potassium conductance, gK(Ca), by its disappearance in a zero-calcium/EGTA medium and its block by cadmium, barium, tetraethylammonium ions, and charybdotoxin. In addition to gK(Ca), a delayed rectifier potassium conductance (gK) is present in or near the presynaptic terminal. Both these potassium conductances are involved in the repolarization of the membrane during a presynaptic action potential.