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(Circulation Research. 1995;76:223-235.)
© 1995 American Heart Association, Inc.

Articles

Propafenone Preferentially Blocks the Rapidly Activating Component of Delayed Rectifier K⁺ Current in Guinea Pig Ventricular Myocytes

Voltage-Independent and Time-Dependent Block of the Slowly Activating Component

Eva Delpón, Carmen Valenzuela, Onésima Pérez, Oscar Casis, Juan Tamargo

From the Department of Pharmacology, School of Medicine, Universidad Complutense, Madrid, Spain.

Correspondence to Eva Delpón, PhD, Department of Pharmacology, School of Medicine, Universidad Complutense, 28040-Madrid, Spain.

	Abstract

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Abstract The effects of propafenone on the delayed rectifier K⁺ current were studied in guinea pig ventricular myocytes by using the patch-clamp technique. In these myocytes, this current consists of at least two components: a La³⁺-sensitive component activating rapidly with moderate depolarizations and a La³⁺-resistant current slowly activating at more positive potentials. In the absence of La³⁺ (when both components are present), propafenone inhibited the delayed outward current, its effects being more marked after weak than after strong depolarizations. Propafenone-induced block of the tail currents elicited on return to -30 mV was more marked after short than after long depolarizing pulses. In the presence of 1 µmol/L propafenone, the envelope-of-tails test was satisfied, thus indicating that at this concentration propafenone completely blocks the rapidly activating component. In the presence of La³⁺ (when only the slow component is present), the steady state inhibition induced by 5 µmol/L propafenone on both the maximum activated and the tail currents was independent of the test pulse voltage. Development of propafenone-induced block on the slowly activating component was very fast and linked to channel opening. In addition, the blockade appeared to be use dependent, with the rate constant of the onset kinetics at 2 Hz being 0.44±0.1 pulse^-1. The recovery process from propafenone-induced block exhibited a time constant of 2.5±0.4 s. These results indicated that propafenone preferentially inhibits the rapidly activating component of the delayed rectifier and that it blocks in a voltage-independent and time-dependent manner the slow component of this current.

Key Words: propafenone • antiarrhythmic drugs • ventricular myocytes • K⁺ currents • patch clamp

	Introduction

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Propafenone is a class I antiarrhythmic agent¹ widely used both for prophylaxis and treatment of cardiac arrhythmias.^{2 3} Electrophysiological studies have demonstrated that propafenone inhibits the maximum upstroke velocity of atrial and ventricular action potentials in a frequency- and voltage-dependent manner^{4 5} and shifts the steady state relation between maximum upstroke velocity and membrane potential toward more negative values.^{6 7} Propafenone binds to the activated state of the Na⁺ channel⁸ and dissociates very slowly from its receptor site; thus, it has been classified as a class Ic antiarrhythmic agent.¹ These pronounced Na⁺ channel–blocking properties result in a marked depression of intracardiac conduction and a prolongation of the QRS complex.^{2 3} In addition, propafenone exhibits ß-adrenergic receptor blocking effects, and very recently, it has been found that it inhibits L-type Ca²⁺ channels in guinea pig ventricular myocytes.⁹

The duration of cardiac action potentials is controlled by a complex interplay of a variety of cardiac ionic currents, eg, inward Na⁺ and L-type Ca²⁺ currents (I_Na and I_Ca, respectively) and outward K⁺ currents. Inhibition of I_Na and I_Ca is expected to shorten the action potential duration (APD) and can explain the observed effects in sheep Purkinje fibers.^{6 7} However, propafenone has also been reported to increase APD in rabbit sinoatrial cells,^{10 11} rabbit¹¹ and guinea pig atrial⁵ and sheep ventricular muscle fibers,⁷ and rabbit Purkinje fibers.¹¹ Clinical studies have demonstrated that propafenone may lengthen ventricular refractoriness, and several cases of drug-induced long QT syndrome with torsade de pointes have been reported in patients with thyroid dysfunction or those treated with amiodarone.^{2 3} Even when the prolongation of refractoriness beyond the APD can be explained as a result of a delay in the recovery of Na⁺ channel from inactivation,¹ the ability of propafenone to prolong the APD can also be explained by a direct effect on the repolarizing outward K⁺ currents. In fact, it has recently been found that propafenone blocks three K⁺ currents (the delayed rectifier current [I_K], the inward rectifier current [I_K1], and the transient outward current [I_to]) in rabbit atrial myocytes¹² and the slowly inactivating delayed rectifier in rat ventricular myocytes.¹³ These K⁺-blocking properties are shared by other members of the so-called Ic antiarrhythmic subclass. In fact, flecainide and encainide also blocked the I_K in cat ventricular myocytes.^{14 15} Moreover, the mechanism of inhibition of K⁺ currents by propafenone, flecainide, and encainide appears to be through open channel block.^{12 13 14} However, the relative importance of the different K⁺ channels may vary among cardiac tissues and animal species and with factors such as heart rate and ischemia.¹⁶ In rabbit and cat myocytes, I_K consists of only one component, which activates at moderate depolarizations and with a fast time course,^{14 17} whereas in guinea pig ventricular myocytes, I_K is composed of a rapidly activating component (I_K,r) and a slowly activating component (I_K,s).¹⁸ It is important to note that the majority of human atrial cells possessed a slowly developing outward current¹⁹ that according to its amplitude and kinetics is more likely to participate in phase 3 of the action potential than is I_to. This current fails to satisfy the envelope-of-tails test¹⁹ and seems to be a composite of a rapidly activating E-4031–sensitive component (I_K,r) and of a slowly activating E-4031–insensitive component (I_K,s).²⁰ In addition, both components share similarities in terms of kinetics, voltage dependence, and rectification properties with the currents described by Sanguinetti and Jurkiewicz¹⁸ in guinea pig ventricular myocytes.

At the present time, there is no information on the effects of propafenone on the slow component of I_K. Therefore, and in order to gain further insight into the possible mechanisms by which propafenone blocked these time-dependent K⁺ channels, in the present study we analyzed its effects on the rapidly and slowly activating components of I_K in single ventricular myocytes from guinea pig hearts.

	Materials and Methods

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Isolation of Cardiac Myocytes
Single ventricular myocytes were isolated from hearts of male guinea pigs (250 to 300 g) by enzymatic dissociation following a procedure previously described in detail.^{21 22 23} Hearts were removed and rapidly mounted via the aorta on the cannula of a Langendorff perfusion system and initially perfused for 1 to 2 minutes with a modified Tyrode's solution containing 1.8 mmol/L CaCl₂. The perfusate was oxygenated (95% O₂/5% CO₂) and maintained at 37°C. Then the hearts were perfused for 5 minutes at 10 to 15 mL/min with a Ca²⁺-free Tyrode's solution, followed by a 4-minute perfusion with the same solution supplemented with 0.12 mg/mL collagenase (Sigma type Ia, Sigma Chemical Co) and 0.03 mg/mL protease (Sigma type XIV). The hearts were then washed with a high-K⁺ low Cl^- solution or KB solution for 4 minutes. Afterward, hearts were removed from the Langendorff apparatus, and the ventricles were dissected and cut in small pieces, which were placed in a beaker containing KB solution and gently shaken to disperse the cells. The resulting cell pellet was stored in KB medium for 1 to 2 hours before beginning the experiments. The composition of the Tyrode's solution was as follows (mmol/L): NaCl 114, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, taurine 20, glucose 10, NaHCO₃ 24, and NaH₂PO₄ 0.42; pH was adjusted to 7.4 by the addition of NaOH. The high-K⁺ low Cl^- solution (KB solution) contained the following (mmol/L): glutamic acid 70, taurine 10, KCl 20, KH₂PO₄ 10, MgCl₂ 1.0, succinic acid 5.0, creatine 5.7, dextrose 10, potassium EGTA 0.2, and potassium HEPES 10; pH was adjusted to 7.4 by addition of KOH.

A small aliquot of a suspension of dissociated cells was transferred to a 0.5-mL chamber placed on the stage of an inverted phase-contrast microscope (model TMS, Nikon Co). After settling to the bottom of the chamber, cells were superfused with standard saline solution at 1 mL/min. To measure the total outward K⁺ current, the external solution contained (mmol/L) NaCl 136, KCl 5.4, CaCl₂ 1.0, MgCl₂ 1.0, CoCl₂ 2.0, tetrodotoxin 0.03, glucose 10, and HEPES 10; the pH was adjusted to 7.4 by addition of NaOH. Under these experimental conditions, I_Na and I_Ca were blocked by tetrodotoxin and CoCl₂, respectively. It has been previously reported that Co²⁺ blocks I_K and shifts the activation curve to a more depolarized voltage range in guinea pig ventricular myocytes perfused in a Na⁺-, K⁺-, and Ca²⁺-free solution.²⁴ However, results obtained in our laboratory when nisoldipine (200 nmol/L) replaced Co²⁺ as I_Ca blocker (in the Na⁺-, K⁺-, and Ca²⁺-containing solution) indicated that both I_K and I_K,s exhibit the same voltage- and time-dependent properties.^{22 23} In the experiments in which the effects of propafenone on I_K,s were studied, the cells were perfused with the same external solution supplemented with 30 µmol/L LaCl₃, which has been described to block rapidly activating K⁺ channels.^{25 26} In the experiments in which the use-dependent effects of propafenone were analyzed, I_K1 was eliminated by removing KCl from the extracellular solution, I_K,r was blocked by the addition of 30 µmol/L LaCl₃, L-type Ca²⁺ channels were blocked by 2 mmol/L CoCl₂, and activation of ATP-sensitive K⁺ channels was prevented by the addition of 3 µmol/L glibenclamide. The pipette solution was composed of (mmol/L) potassium aspartate 80, KCl 42, KH₂PO₄ 10, magnesium ATP 5, phosphocreatine 3, potassium EGTA 5, and potassium HEPES 5. The pH was adjusted to 7.2 with KOH. Junction potentials between the pipette and external solution were -9 mV. This potential was offset before the initiation of each experiment. All experiments were performed at room temperature (22°C to 23°C) on Ca²⁺-tolerant healthy ventricular myocytes (80 to 110 µm in length) identified by their clear rodlike striated appearance and a lack of any visible bleb on the surface.

Recording Techniques
Membrane currents were measured with the perforated–nystatin patch configuration of the patch-clamp technique²⁷ by using an Axopatch-1D clamp amplifier (Axon Instruments). Micropipettes were constructed from borosilicate capillary tubes (model GD-1, Narishige Co, Ltd) by using a programmable patch micropipette puller (model P-87 Brown-Flaming, Sutter Instruments Co). The pipettes had resistances of 2 to 4 M when filled with the internal solution and immersed in the external solution. Series resistance was compensated manually by using the series resistance compensation unit of the Axopatch amplifier, and 80% compensation was achieved. The effective access resistance was calculated from the ratio of _a to C_m, where _a is the time constant of uncompensated capacitive transient and C_m is the cell capacitance obtained by integration of uncompensated capacitive transients, as previously described by Sala et al,²⁸ and the values obtained averaged 13.0±0.9 M (n=8). Since the series resistance was compensated by 80% and the bigger currents recorded were <1 nA (633.3±50.2 pA), the mean value of voltage error has an upper limit of 2.6 mV. During the experiments, membrane potential and current data were displayed on a storage oscilloscope (model 5020A, Kikusui Electronics Corp). Voltage-clamp command pulses were generated by a 12-bit digital-to-analog converter. Membrane currents were filtered at 1 kHz (-3 dB) by a four-pole Bessel filter before sampling was done at 2 kHz by a 12-bit analog-to-digital converter. A Tandon 386/25 computer with PCLAMP 5.5.1 software (Axon Instruments) was used to generate voltage-pulse protocols and to acquire and analyze data. In experiments in which the use-dependent effects of propafenone were analyzed, currents during trains of pulses were also displayed by a recorder (model 220, Gould Inc).

The choice of the nystatin method in the present experiments was guided by previous studies using nystatin to permeabilize cardiac myocytes and record I_Ca and I_K without washout of the intracellular media.^{22 23} Under these experimental situations, intracellular conditions appeared unaltered, avoiding the "rundown" that is known to occur for I_K and particularly for its slow component (I_K,s) with the normal whole-cell recording.¹⁶ In fact, it has been demonstrated that the total I_K in guinea pig ventricular myocytes remained unaltered for up to 70 minutes after starting the measurements when using this configuration of the patch-clamp technique.^{22 23} In the present experiments, after a 15-minute period for control measurements, the perfusate was changed to one containing propafenone hydrochloride, and data were collected again after 10 minutes. At this time, cells were perfused again with drug-free solution to determine the reversibility of drug action. The inhibition produced by propafenone on I_K was almost completely reversible on washout, with the time course of this process being very slow, as previously described by Duan et al.¹²

Voltage and Time Dependence
Steady state voltage-dependent drug effects were evaluated in two ways. The relation between transmembrane voltage and steady state current was assessed directly by clamping from -40 mV to several depolarizing test potentials for 5 seconds. The maximum outward current developing during a positive clamp step was measured for increasing test potentials from -30 to +70 mV as the difference between the instantaneous current level after the decay of the capacity transient and the current level at the end of the voltage step (I_K,max). A second measure of I_K activation was obtained by clamping to -30 mV immediately after the 5-second clamp step at the depolarizing potential. The amplitude of the deactivating current tails was measured as the difference between the maximum outward current immediately after the step to -30 mV (after the inward capacity artifact) and the steady state current measured after 5 seconds at -30 mV (I_K,tail). Intervals between successive pulse protocols were 45 to 60 s to avoid residual activation of I_K.

Time-dependent drug effects were quantified by measuring the amplitudes of tail currents elicited by depolarization pulses from -40 to +30 mV of variable durations (from 0.1 to 10 s). The relation between the amplitude tails and the pulse duration was fitted by a monoexponential time function.

Data Analysis and Curve Fitting
Data obtained under control conditions were compared with those obtained after drug exposure in a paired manner. For comparisons at a single voltage or time point, differences were analyzed by Student's t test. To analyze block at multiple voltages or drug concentrations, two-way ANOVA was used.²⁹ All data are presented as mean±SEM.

I_K activation curves were fitted by a Boltzmann distribution using a least-squares fitting routine:

where V_h is the half point of activation (in millivolts), V_m is the test potential, and k represents the slope factor for the activation curve (in millivolts).

The activation kinetics of I_K,s has been described as a sigmoidal process, assuming that the channel has multiple closed states.²⁵ However, in the present study and in order to describe the dominant time constant of this process and the effects of propafenone on it, an exponential analysis was used as an operational approach, fitting the activation and the deactivation kinetics to an equation of the following form:

where ₁, ₂, and ₃ are the system time constants; A₁, A₂, and A₃ are the amplitudes of each component of the exponential; and C is the baseline value. The curve-fitting procedure used a nonlinear least-squares (Gauss-Newton) algorithm. Fits were displayed in a linear and semilogarithmic format together with the difference plot. Goodness of fit and required number of exponential components were judged by comparing ² values statistically (F test). In addition, we looked for nonrandom deviations in the difference plot.³⁰ The same operational approach was used to analyze the time constant of development and the time constant of the recovery of propafenone-induced block.

Chemicals
Chemicals were obtained from Sigma Chemical Co. Propafenone hydrochloride (Knoll AG) as a powder was dissolved in deionized water to make a 1-mmol/L stock solution. Further dilutions were performed in Tyrode's solution to give the desired final concentration immediately before each experiment. Glibenclamide (Sigma) and dofetilide (Pfizer) were prepared as a 10-mmol/L stock solution in dimethyl sulfoxide. At the final concentration used, dimethyl sulfoxide had no effect on K⁺ currents.³¹

	Results

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Effects of Propafenone on Total I_K
Fig 1 shows examples of the effects of propafenone at 0.5 µmol/L (panel A) and 5 µmol/L (panel B) on I_K in two different cells. Membrane outward currents were elicited by applying 5-s depolarizing pulses from holding potentials of -40 to +30 mV, followed by a repolarization of 5 s to -30 mV. Pulses were applied every 30 s. Depolarizing pulses evoked a slowly rising outward current and, on repolarization to -30 mV, an outward tail current (I_K,tail), which has been described as the deactivation of the delayed rectifier.³² I_K,max, activated by the depolarizing pulse, was measured as the difference between the instantaneous current level after the decay of the capacity transient and the current level at the end of the voltage step. Under control conditions, the amplitude of I_K,max and I_K,tail averaged 326±24 and 185±11 pA, respectively (n=22). Propafenone inhibited both I_K,max and I_K,tail in a concentration-dependent manner. Thus, in the presence of 0.5, 1, and 5 µmol/L, propafenone decreased I_K,max at +30 mV by 16.7±5.9% (n=6), 30.2±13.3% (n=8), and 41.6±4.9% (n=8) and I_K,tail by 23.7±2.6% (n=6), 32.6±2.8% (n=8), and 52.0±5.3% (n=8), respectively (P<.05 versus control for all doses). These results indicated that at these three concentrations, propafenone produced a similar amount of block on I_K,max and I_K,tail (P>.05). The inhibition of I_K produced by propafenone was partially reversible on washout, but the time course of this process was very slow, as previously described by Duan et al.¹² The lower current tracings in both panels of Fig 1 (designated S) represent the propafenone-sensitive current obtained by digital subtraction of the two tracings. At the lower concentration (0.5 µmol/L), the propafenone-sensitive current reached steady state in <2 s. In contrast, at 5 µmol/L, the current blocked by propafenone progressively increases over the time of the application of the depolarizing pulse. Fig 1 also shows that at the lower concentration, the propafenone-sensitive current exhibits a tail current as large as the maximum activated current during the depolarizing step. In contrast, the tail current of the 5-µmol/L propafenone–sensitive current was smaller than the current activated during the depolarization.

View larger version (16K): [in this window] [in a new window]   Figure 1. Tracings showing the effects of 0.5 µmol/L propafenone (A) and 5 µmol/L propafenone (B) on outward K+ current in two different cells. Currents were recorded during 5-s pulses to +30 mV from a holding potential of -40 mV before (C) and after exposure to propafenone (P). The propafenone-sensitive current (S) was obtained by digital subtraction between the two tracings (control and propafenone). The dotted line represents the zero current level.

Envelope-of-Tails Test
I_K in guinea pig ventricular myocytes is due to the activation of two components, I_K,r and I_K,s.¹⁸ I_K,r activates rapidly with moderate depolarizations (between -40 and 0 mV), whereas I_K,s activates slowly with a sigmoidal time course at more positive potentials. To test whether propafenone preferentially blocks one or both components of the aggregate outward current (I_K), in the next group of experiments, the relation between propafenone-induced block and the duration of the depolarizing pulse was studied. Fig 2 shows typical membrane recordings elicited by pulses of 0.1, 0.4, 0.7, 1, 2, and 4 s from -40 to +30 mV in the same cell before (left panel) and after exposure to 5 µmol/L propafenone (right panel), respectively. It can be observed that propafenone blocks I_K,tail, but its inhibitory effect was greater for short than for long pulses. In fact, under control conditions after a 0.1-s depolarizing pulse to +30 mV, a clearly identifiable I_K,tail was elicited, whereas no I_K,tail was observed in the presence of propafenone. In this experiment, the amplitude of I_K,tail was blocked by 53% (from 49 to 23 pA) after a depolarizing pulse of 0.4 s but only by 33% (from 147 to 98 pA) after a 4-s depolarizing pulse, respectively. Similar results were obtained in five other cells (Table).

View larger version (13K): [in this window] [in a new window]   Figure 2. Tracings showing the envelope-of-tails test for delayed rectifier K+ current before and after the addition of 5 µmol/L propafenone. The external solution contained 2 mmol/L CoCl2. Membrane potential was held at -40 mV, and pulses to +30 mV for variable periods (0.1, 0.4, 0.7, 1, 2, and 4 s) were applied every 30 s in the absence (left) and presence (right) of propafenone. The dotted line represents the zero current level.

View this table: [in this window] [in a new window]   Table 1. Percentage of Inhibition of the Delayed Rectifier K+ Current and Its Slowly Activating Component Induced by Propafenone (5 µmol/L) Measured on Tail Currents Elicited on Return to -30 mV After 0.4-, 4-, and 10-s Depolarizing Pulses From -40 to +30 mV

These results suggest an inhibitory effect of the drug on I_K,r, since it has been described that this component activates rapidly with moderate depolarizations. To further analyze this hypothesis, the envelope-of-tails test was performed. This test predicts that if I_K represents the activation of a single type of channel, then the magnitude of I_K,tail after a given depolarizing pulse of variable duration should increase parallel to the outward current during the depolarizing pulse.¹⁸ Therefore, given one channel type, the ratio of tail current to time-dependent current (I_K,tail/I_K,max) should be constant, regardless of the duration of the pulse. The results of such a test on 12 cells are shown in Fig 3, where I_K,tail/I_K,max is represented as a function of the test pulse duration. Test pulses were applied from a holding potential of -40 to +30 mV for durations ranging from 0.1 to 10 s. In cells perfused with Co²⁺-containing solution, the ratio was dependent on the duration of the pulse. Thus, as the duration of the pulse was lengthened, the magnitude of I_K,tail became smaller than that of I_K,max measured during the test pulses to +30 mV; thus, for pulses 1 s I_K,tail/I_K,max of 0.54±0.06 (n=6) was obtained. These results agree with previous reports^{18 22 25} that demonstrate the existence of two different components of I_K in guinea pig ventricular myocytes as the result of the activation of two different K⁺ channel types, a rapidly and a slowly activating channel. In the presence of 0.5 µmol/L propafenone (Fig 3A), there was a significant reduction of I_K,tail/I_K,max for depolarizing pulses shorter than 1 s (from 0.96±0.1 to 0.73±0.1 for pulses of 0.1 s and from 0.76±0.07 to 0.51±0.02 for pulses of 0.85 s, n=6, P<.05), but it was clear that the remaining (drug-insensitive) outward current was still a composite of two channel types. After exposure to 1 µmol/L propafenone (Fig 3B), I_K,r was blocked, and the remaining outward current was composed of a single current type. In fact, at this concentration of propafenone, only the I_K,s component is present and I_K,tail/I_K,max remained constant at 0.45±0.005 regardless of the duration of the test pulse. This value is quite close to the predicted one (0.48) from the ratio of the driving force at +30 mV and at -30 mV for a nonrectifying K⁺-selective outward current. Therefore, these results strongly suggest that propafenone blocks, in a concentration-dependent manner, the rapidly activating component of I_K.

View larger version (15K): [in this window] [in a new window]   Figure 3. Graphs showing the envelope-of-tails test as a ratio between tail current (IK,tail) and time-dependent current (IK,max) elicited by test pulses applied from a holding potential of -40 mV to +30 mV for durations ranging from 0.1 to 10 s followed by repolarization to -30 mV. IK,tail/IK,max was plotted as a function of the pulse duration in both the absence () and presence () of 0.5 µmol/L (A) or 1 µmol/L (B) propafenone. Values represent the means, and vertical lines indicate SEM of 12 cells.

To further study this possibility, the effects of 5 µmol/L propafenone on I_K,max and I_K,tail were analyzed in response to 5-s depolarizing pulses from a holding potential of -40 mV to various test potentials (from -30 to +70 mV) followed by repolarization to -30 mV for 5 s. During test pulses to potentials >-20 mV, propafenone significantly reduced I_K,max, this reduction being more marked after weak than after strong depolarizing pulses. In six cells, the percentage of block of I_K,max after pulses to 0 and +70 mV averaged 54.4±5.0% (from 123±21 to 54±7 pA, P<.05) and 33.6±5.2% (from 500±87 to 318±38 pA, P<.05), respectively. Furthermore, in the presence of propafenone (5 µmol/L), the amplitude of I_K,tail decreased at all potentials tested, with the blockade being less pronounced with greater depolarizations. Thus, in six cells, propafenone reduced by 67.6±6.3% (from 69±10 to 21±4 pA, P<.05) I_K,tail obtained on return to -30 mV after a 5-s pulse to 0 mV and by 47.1±4.5% (from 264±42 to 134±16 pA, P<.05) I_K,tail after pulses to +70 mV.

Effects of Propafenone on I_K,s
Although at lower concentrations propafenone blocks I_K,r, the previous data shown suggest that it also blocks I_K,s to a certain extent. To determine the effects of propafenone on I_K,s, currents were measured in cells perfused with 30 µmol/L La³⁺–containing external solution. At this concentration, La³⁺ completely blocks I_K,r, thereby permitting the specific evaluation of propafenone effects on I_K,s.²⁶ Fig 4A shows current tracings obtained by applying 5-s depolarizing pulses to +30 mV from a holding potential of -40 mV in the presence of 30 µmol/L La³⁺ and when the external solution was supplemented by 100 µmol/L dofetilide, a specific blocker of I_K,r.^{31 33} At this high concentration, dofetilide did not modify the remaining current in the presence of La³⁺. To further examine the effects of La³⁺ and dofetilide on K⁺ currents, an envelope-of-tails test was performed in the same cell. The results of such a test are shown in Fig 4B, where I_K,tail/I_K,max is represented as a function of the test-pulse duration. Test pulses were applied from a holding potential of -40 to +50 mV for durations ranging from 0.25 to 7 s. It was evident that, in the absence of La³⁺ and dofetilide, I_K was the composite of I_K,r and I_K,s. In contrast, in the presence of 30 µmol/L La³⁺ alone or in the presence of La³⁺ plus 100 µmol/L dofetilide, I_K,r was absent, and the remaining current was due to the activation of I_K,s. The same results were obtained in two other cells. These results indicated that 30 µmol/L La³⁺ completely and rapidly blocks I_K,r.

View larger version (16K): [in this window] [in a new window]   Figure 4. A, Current tracings recorded in the absence and presence of 100 µmol/L dofetilide. The holding potential was maintained at -40 mV, and 5-s depolarizing pulses were applied to +30 mV. The external solution contained 30 µmol/L LaCl3. The dotted line represents the zero current level. B, The ratio of tail current to time-dependent current (IK,tail/IK,max) plotted as a function of the test pulse duration. Test pulses were applied from a holding potential of -40 to +50 mV for durations ranging from 0.25 to 7 s. indicates IK,tail/IK,max when the cell was perfused with 2 mmol/L Co2+–containing solution; , data obtained in the presence of 30 µmol/L La3+; and , IK,tail/IK,max when the solution was supplemented with 100 µmol/L dofetilide.

Fig 5A shows examples of currents measured before and after exposure to 5 µmol/L propafenone in the presence of 30 µmol/L La³⁺. Outward currents were elicited by 5-s depolarizing pulses from a holding potential of -40 to +30 mV, followed by a repolarization of 5 s to -30 mV. The maximum activated current (I_Ks,max) was measured as the difference between the instantaneous current level after the decay of the capacity transient and the current level at the end of the voltage step. The lower current tracing represents the propafenone-sensitive current obtained by digital subtraction. In this experiment, propafenone decreased I_Ks,max from 285 to 203 pA. Under these experimental conditions, in the six cells examined, the inhibition of I_Ks,max produced by 5 µmol/L propafenone averaged 30.3±2.9% (from 302±70 to 210±48 pA, P<.05). The current ratio between the propafenone-sensitive current during the depolarizing pulse and the current in control conditions [ie, (I_C-I_P)/I_C] is shown in Fig 5B. An initial value of 0.0 in this figure indicates no block. As can be observed, the block increases during the application of the depolarizing pulse, reaching steady state within the first second. To determine the major dominant time constant of development of block, the onset of block was fitted by a single exponential function, as shown by the solid curve in the figure. The time constant of development of block in six cells averaged 432±47.7 ms when 5-s depolarizing pulses to +30 mV were applied. The same experimental approach was performed at another two test potentials, +10 and +50 mV. In six cells, the time constant of development of block at +10 mV averaged 943.8±36.9 ms, and it averaged 291.1±85.9 ms when the depolarizing pulses were applied to +50 mV (P<.01). These results indicated that the kinetics of the development of block was voltage dependent, being significantly faster at more depolarized potentials. In contrast, the reduction of I_K,s measured at the end of the 5-s depolarizing pulses was similar at different potentials tested (from -10 to +70 mV), and no statistical differences were obtained. Thus, 5 µmol/L propafenone reduced I_Ks,max elicited by depolarizing pulses to +10 mV by 36.9±4.7% (from 124.9±35.8 to 84.0±25.1 pA) and by 30.8±3.3% (from 497.6±98.7 to 348.4±70.8 pA) after pulses to +50 mV (P>.05).

View larger version (17K): [in this window] [in a new window]   Figure 5. A, Current tracings recorded in the absence and presence of 5 µmol/L propafenone. The holding potential was maintained at -40 mV, and 5-s depolarizing pulses were applied to +30 mV. The external solution contained 30 µmol/L LaCl3. The propafenone-sensitive current (subtraction) was obtained by digital subtraction. The dotted line represents the zero current level. B, Plot showing the current ratio between the propafenone-sensitive current during the depolarizing pulse and the maximum outward current in control conditions [ie, (IC-IP)/IC]. An initial value of 0.0 in this figure indicates no block. The continuous line represents the monoexponential function that fits the experimental data.

In another group of experiments, 10 µmol/L propafenone decreased I_Ks,max by 36.1±7.0% (from 206±29 to 131±29 pA, P<.05, n=6). In the presence of this concentration of propafenone, the ratio of current tracings during the depolarizing pulse also increases, and the time constant of block development averaged 423±30.1 ms when 5-s depolarizing pulses were applied to +30 mV.

Voltage-Dependent Effects of Propafenone on I_K,s
The voltage dependence of I_K,s block induced by propafenone was examined further by analyzing I_K,tails elicited on return to -30 mV after 5-s depolarizations to various test potentials in the cells perfused with 30 µmol/L La³⁺. Fig 6A shows typical recordings elicited on return to -30 mV after 5-s depolarizing pulses from -40 to +10, +30, +50, and +70 mV before and after exposure of the cell to 5 µmol/L propafenone. After exposure to the drug, I_K,tail was reduced at all test pulses. Under control conditions, the amplitude of I_K,tail recorded at -30 mV after a 5-s depolarizing pulse from -40 mV to +30 mV averaged 152.8±28.5 pA (n=14), and exposure to propafenone (5 and 10 µmol/L) reduced its amplitude by 36.8±1.6% and 46.2±3.6%, respectively. Fig 6B shows the I_K,s activation curve in the absence and presence of 5 µmol/L propafenone. The activation curve was obtained by normalizing the tail amplitude at each potential against the maximum I_K,tail amplitude for each experiment. The discontinuous line represents the activation curve in the presence of propafenone normalized to the maximum I_K,tails recorded in the presence of the drug. Under control conditions, the activation midpoint averaged 29.9±2.7 mV, and the slope factor averaged 16.3±2.6 mV (n=8). In six cells, 5 µmol/L propafenone did not modify either the midpoint (30.6±2.5 mV, P>.05) or the slope factor (16.1±0.6 mV, P>.05). In six other cells, 10 µmol/L propafenone also failed to modify either the midpoint (37.0±1.0 versus 36.5±1.0 mV) or the slope factor (17.0±1.0 versus 16.4±0.7 mV) of the activation curve.

View larger version (11K): [in this window] [in a new window]   Figure 6. Voltage-dependent effects of propafenone on the slowly activating component of the delayed rectifier K+ current (IK,s). A, Superimposed tail current (IK,tail) tracings elicited on return to -30 mV (arrow) after 5-s depolarizing pulses from a holding potential of -40 mV to +10, +30, +50, and +70 mV before (left) and after (right) exposure to 5 µmol/L propafenone. The dotted line represents the zero current level. B, Graph showing voltage dependence of IK,s activation, measured as the peak IK,tails at -30 mV vs the previous depolarizing potential in the absence () and presence () of 5 µmol/L propafenone. The continuous line represents the best fit obtained using a Boltzmann equation: IK/IKmax=1/{1+exp[(Vh-Vm)/k]}, where IK is outward K+ current, IKmax is maximal IK, Vh is the half point of activation, Vm is the test potential, and k is the slope factor. The discontinuous line represents the activation curve in the presence of propafenone normalized to the maximum IK,tails recorded in the presence of the drug. Values represent the mean±SEM of eight cells.

The results indicated that there were no differences in the amount of block at the different potentials tested, so the steady state inhibition of I_Ks,max and I_K,tail induced by propafenone appears to be independent of test-pulse voltage.

Effects of Propafenone on Activation Kinetics of I_K,s
The effects of 5 µmol/L propafenone on the activation kinetics of I_K,s were studied in eight cells perfused with 30 µmol/L La³⁺–containing solution. Fig 7 shows the tail amplitudes at -30 mV after depolarizing pulses from -40 to +30 mV of increasing duration (0.25 to 10 s) applied every 30 s in the absence and presence of propafenone. Under these experimental conditions, the activation kinetics was fitted as a monoexponential function to determine the major dominant time constant before and after drug exposure (see "Materials and Methods"). The time constant of the activation kinetics in eight cells averaged 3.5±0.3 s in control conditions and 3.3±0.2 s in the presence of propafenone, indicating a lack of drug effect on activation kinetics of I_K,s. Similar results were obtained in six cells exposed to 10 µmol/L propafenone (3.6±0.2 versus 3.3±0.1 s, P>.05). Fractional I_K,tail block as a function of pulse duration is also shown in Fig 7 (squares). The fractional block is observed to increase with the time of depolarization; this process is very fast within the first second. However, for pulses <0.7 s, the amplitudes of I_K,tail were very small, making reliable analysis difficult. The Table shows the percentage of inhibition of I_K,s induced by propafenone measured on I_K,tails elicited on return to -30 mV after 0.4-, 4-, and 10-s depolarizing pulses. The percentage of block tends to increase with the duration of the depolarizing pulse, but this increase did not reach statistically significant values.

View larger version (19K): [in this window] [in a new window]   Figure 7. Graph showing the effects of propafenone on activation kinetics of the slowly activating component of the delayed rectifier K+ current (IK,s). Amplitude of the tail current (IK,tail) obtained in eight cells, in the presence of 30 µmol/L La3+, on return to -30 mV after a depolarizing pulse to +30 mV of variable duration (0.25 to 10 s) was plotted against the duration of the pulse in the absence () and presence () of 5 µmol/L propafenone. The continuous line represents the monoexponential function that fits the experimental data. indicates the fractional block of IK,tails [(IC-IP)/IC], where IC indicates IK,tails in control conditions and IP represents IK,tails in the presence of 5 µmol/L propafenone] obtained with the same experimental protocol as a function of the duration of the depolarizing pulse. Values represent the means, and vertical lines indicate SEM of eight cells.

Time-dependent interactions of propafenone on I_K,s were also studied by analyzing its effects on the deactivation time course. Under control conditions, the deactivation kinetics of I_K,tails elicited on return to -30 mV after 5-s depolarizing pulses from -40 to +30 mV were fitted by a biexponential function. In eight cells, 5 µmol/L propafenone did not modify either the fast (326.1±31.4 versus 335.1±30.1 ms, P>.05) or the slow (1611.2±328.7 versus 2004.2±376 ms, P>.05) time constants of deactivation. Exposure to 10 µmol/L propafenone also failed to produce any significant change on the deactivation kinetics (fast time constant, 217.3±62.8 versus 302.5±93.9 ms; slow time constant, 1635.2±226.1 versus 2230.7±376.2 ms; P>.05; n=6).

Use-Dependent Effects of Propafenone
To further characterize the effects of propafenone on I_K,s, in the next group of experiments the use-dependent effects of propafenone were studied. In these experiments, I_K1 was eliminated by removing KCl from the extracellular solution, I_K,r was blocked by addition of 30 µmol/L LaCl₃, I_Ca through L-type Ca²⁺ channels was blocked by 2 mmol/L CoCl₂, and activation of ATP-sensitive K⁺ channels was prevented by the addition of 3 µmol/L glibenclamide (see "Materials and Methods"). Under these conditions, only I_K,s and K⁺ current with high activity at plateau potentials (I_Kp)³⁴ were activated during test depolarizations, as previously described by Jurkiewicz and Sanguinetti.³¹ Fig 8A, left, shows membrane current tracings obtained by applying 1-s depolarizing pulses from -40 to +50 mV every 60 s before and after exposure to 5 µmol/L propafenone. Fig 8A, right, shows the current tracings recorded in the same cell when the same stimulation protocol was preceded by a train of 50 depolarizing pulses of 200-ms duration from -40 to +50 mV with an interpulse interval of 330 ms. A rest period of 3 minutes separated the trains. Fig 8B shows original recordings obtained in a different cell, when the conditioning train was applied in the absence and presence of propafenone (5 µmol/L). Under control conditions, there is a use-dependent increase in outward current. In four cells, the amplitude of both I_Ks,max and I_K,tail measured at the test pulse of +50 mV after conditioning pulse trains at 3 Hz was 1.5±0.1 and 1.7±0.06 times larger, respectively, than those obtained when only a single test pulse was applied. This use-dependent increase has been attributed to an incomplete deactivation of I_K,s during the brief time (330 ms in our experiments) that the membrane potential was held at -40 mV when conditioning pulses were applied.³¹ In the experiment shown in Fig 8A, 5 µmol/L propafenone reduced I_Ks,max and I_K,tail by 20.4% (from 368 to 293 pA) and by 23.8% (from 201 to 153 pA), respectively, when a single pulse was applied. When the same cell was stimulated previously by a train of pulses, the reduction on I_Ks,max and I_K,tail observed when applying a 1-s depolarizing pulse to +50 mV was 19.6% (from 607 to 488 pA) and 21.1% (from 350 to 276 pA), respectively. Thus, under these experimental conditions, the blockade of I_K,s produced by 5 µmol/L propafenone, measured on the maximum activated current after 1-s test pulse, was not significantly different when the cell was stimulated previously by a train of pulses than when a depolarizing pulse was applied every 60 s under rest conditions (21.9±3.2% versus 20.8±1.6%, n=4, P>.05). The block induced by propafenone on I_K,tail was also similar when the pulse was preceded and not preceded by a train of pulses (33.3±3.5% versus 26.9±3.4%, n=4, P>.05). Visual inspection of the current recordings of Fig 8B revealed that in the presence of 5 µmol/L propafenone, I_Ks,max and I_K,tail show summation with repetition of the clamp depolarization and that this phenomenon was not reversed in the presence of the drug. However, since at +50 mV the block develops very quickly, measurement of block induced by propafenone during the 1-s depolarizing pulse may reflect essentially the block developed during the pulse itself and not the modification of the amount of block during the application of the train of pulses.

View larger version (64K): [in this window] [in a new window]   Figure 8. Use dependence of the effects of propafenone on the slowly activating component of the delayed rectifier K+ current (IK,s). A, Current tracings recorded before and after exposure to 5 µmol/L propafenone when applying a 1-s test pulse from -40 to +50 mV (left) or when the same stimulation protocol was preceded by a train of 50 depolarizing pulses of 200-ms duration from -40 to +50 mV with an interpulse interval of 330 ms (right). The dotted line represents the zero current level. B, Current recordings obtained in a different cell in control conditions (top) and in the presence of 5 µmol/L propafenone (bottom). Depolarizing pulses 1 s in duration were applied to +50 mV, from a holding potential of -40 mV, after conditioning trains of 50 depolarizing pulses from -40 to +50 mV (200 ms in duration) at a frequency of 3 Hz.

To assess whether the change in pulse interval can modify the total amount of I_K,s block, in another series of experiments trains of 16 short (200-ms) depolarizing pulses to +50 mV from a holding potential of -40 mV were applied at a repetitive frequency of 0.5, 1, and 2 Hz. Outward currents elicited during the depolarizing pulse were measured immediately after the capacitive transient. The results obtained in a typical experiment when a train at 2 Hz was applied in the absence and presence of 5 µmol/L propafenone are shown in Fig 9A. Under control conditions, the outward currents during the depolarizing step and I_K,tails increased during the application of the train, and this effect was more marked as the frequency increased. The percentage of increase of the outward current elicited by the depolarizing step from the first to the last pulse was 62.8±23.5%, 86.4±43.3%, and 199.6±59.5% for trains at 0.5, 1, and 2 Hz, respectively. In the presence of 5 µmol/L propafenone, the outward current also increased during the trains of pulses, but this increase was less marked than that observed under control conditions, even when the difference reached statistical significance only at 2 Hz (138.3±50.2%, n=4, P<.05).

View larger version (23K): [in this window] [in a new window]   Figure 9. A, Graph showing outward current (circles) and tail current (triangles) amplitudes in control conditions (solid symbols) and in the presence of 5 µmol/L propafenone (open symbols) during trains of 16 depolarizing pulses (200 ms in duration) from -40 to +50 mV at a frequency of 2 Hz. IK,s indicates the slowly activating component of the delayed rectifier K+ current. Outward currents elicited during the depolarizing pulse were measured immediately after capacitive transient. B, Graph showing the ratio of the amplitude of the outward current () and the tail currents () in the presence (IP) and in the absence of propafenone (IC) plotted as a function of the number of pulses. The continuous line represents the monoexponential function that fits the IP/IC ratio for the outward current against the consecutive pulses of the train.

Fig 9B shows the ratio of the amplitude of the outward current in the presence (I_P) and in the absence (I_C) of propafenone as a function of the number of pulses. The blockade induced by 5 µmol/L propafenone increased during the application of trains at 2 Hz, reaching a final value of 18.2±3.4% (n=4). The onset kinetics of the use-dependent block at 2 Hz was analyzed by fitting to a monoexponential function the relative current (I_P/I_C) against the number of consecutive pulses of the train. When this procedure was used, the rate constant of the onset kinetics of block averaged 0.44±0.1 pulse^-1.

Similar results were obtained when I_K,tails were analyzed (Fig 9). However, as a consequence of the fast development of block at depolarized levels, a certain amount of block appeared within the first pulse of the train (11.9±3.4%), an effect that was independent of the frequency of stimulation. The block of I_K,tails increased to 19.2±0.3% and 23.6±1.7% at the end of depolarizing trains at 1 and 2 Hz, respectively.

Recovery From Propafenone-Induced Block on I_K,s
Recovery from propafenone-induced I_K,s block was measured by using a double-pulse protocol in which a conditioning prepulse from -40 to +50 mV for 3 s, which was enough to fully develop the steady state blockade, was followed by a 1-s test pulse of the same amplitude. The coupling interval between both pulses was progressively increased from 0.3 to 10 s. Fig 10A shows current tracings recorded in control conditions and in the presence of 5 µmol/L propafenone for three coupling intervals (0.3, 1, and 7.5 s). At shorter coupling intervals, I_Ks,max and I_K,tail amplitudes were larger. Fig 10B shows the amplitude of the outward current elicited by the test pulse as a function of the time interval for the experiment shown in Fig 10A. Currents were measured again immediately after the capacitive transient to avoid the blockade developed during the 1-s depolarizing pulse itself. As can be observed, current amplitude decreased as the time interval between the pulses increased, and this decline can be adjusted to a monoexponential function. The time constant of this decline averaged 1306.4±172.7 ms under control conditions and 1202.9±165.8 ms in the presence of 5 µmol/L propafenone (P>.05, n=12), respectively. The amplitude of the initial current compared with the control (ie, initial current ratio) can be considered as a measure of the recovery from block occurring at the holding potential.³⁵ Fig 10C shows the ratio of the current amplitude at each coupling interval for this particular experiment. In the present experiments, there was a delay at the beginning of the recovery. Thus, to calculate the dominant time constant of recovery the experimental values were fitted by a monoexponential function after exclusion of the first data points. The time constant of recovery calculated by using this procedure averaged 2.5±0.4 s (n=12).

View larger version (20K): [in this window] [in a new window]   Figure 10. Time-dependent inhibition of the slowly activating component of the delayed rectifier K+ current (IK,s). A, Currents recorded in the absence (top) and presence (bottom) of 5 µmol/L propafenone. A double-pulse protocol was applied. A 3-s conditioning prepulse from -40 to +50 mV was followed by a test pulse of 1-s duration of the same amplitude at various rest intervals (from 0.3 to 10 s). The current tracings shown in this figure were obtained at pulse intervals of 0.3, 1, and 7.5 s. Note that at shorter coupling intervals, the maximum outward and tail current amplitudes were larger. B, Graph showing the amplitude of outward currents elicited by the test pulse as a function of the time interval in the absence () and presence () of 5 µmol/L propafenone. Data were obtained from the same cell as in panel A. Continuous lines represent the monoexponential fit of the decline of the currents as a function of the interstimulus interval. C, Graph showing the amplitude of the initial current compared with control (IP/IC) as a function of the time interval. Experimental values were fitted by a monoexponential function (solid line) after exclusion of the first data points to calculate the dominant time constant of recovery.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present article, we have studied the effects of propafenone, a potent open Na⁺ channel blocker with slow kinetics,^{1 8} on the two components of I_K (I_K,r and I_K,s) in guinea pig ventricular myocytes. The two components can be distinguished by their activation kinetics, rectification, and drug sensitivity.^{18 25 26} I_K,r rapidly activates at moderate depolarizations (between -40 and 0 mV), and the fully activated current-voltage relation shows inward rectification at potentials >10 mV and can be blocked by E-4031¹⁸ and dofetilide.^{31 33} I_K,s activates more slowly but is much larger than I_K,r when fully activated and becomes manifest only at depolarizations positive to 0 mV, rectification is minimal or absent, and I_K,s is insensitive to drugs that fully block I_K,r.¹⁸

Effects of Propafenone on I_K
The present results show that propafenone blocks total I_K in a concentration-dependent manner, with the blockade being more marked after weak than after strong depolarizing pulses both on I_Kmax and on I_K,tail obtained on return to -30 mV. In addition, the effects of propafenone on total I_K were also time dependent, so that it significantly induced more block after short (0.1 to 1 s) than after long depolarizing pulses. Since I_K,r activates at more negative potentials and with faster kinetics than I_K,s,¹⁸ the effects of propafenone on total I_K may suggest that it preferentially blocks the rapidly activating component, I_K,r.

This hypothesis was confirmed when the envelope-of-tails test was performed. Given a single channel type, the time course of the envelope-of-tail current amplitudes plotted as a function of voltage pulse duration should parallel the amplitude of the time-dependent current during the pulse. As previously reported,^{18 25} in guinea pig ventricular myocytes, I_K appears to be a composite of at least two different channel types. Propafenone inhibited in a concentration-dependent manner I_K,tail/I_K,max for depolarizing pulses ranging from 0.1 to 1 s, so that at 1 µmol/L the drug completely inhibited I_K,r. These results indicated that propafenone was a very potent blocker of the rapidly activating K⁺ channels and confirmed previous results obtained in rabbit atrial myocytes,¹² where I_K consisted of only one component (I_K,r), which developed for moderate depolarizations and with a fast time course.¹⁷ Very recently, Duan et al¹² demonstrated that propafenone blocks I_K,r by binding preferentially to an open state of the channel, but in the present experimental conditions, we were not able to determine the possible voltage and time dependence of the inhibition of I_K,r induced by propafenone.

Effects of Propafenone on I_K,s
To assess the effects of propafenone on I_K,s, cells were perfused with 30 µmol/L La³⁺–containing solution. At this concentration, La³⁺ completely blocks I_K,r.²⁶ We also performed a control test by adding a high concentration of dofetilide (100 µmol/L) in the presence of 30 µmol/L La³⁺. As shown in Fig 4, dofetilide did not significantly modify the remaining outward current in the presence of La³⁺, and the currents recorded both in the presence of La³⁺ alone or when the solution was supplemented with dofetilide satisfied the envelope-of-tails test, thus indicating that only the slow component of the delayed rectifier was present. It has been previously reported that La³⁺ at concentrations of >10 µmol/L screens and binds negative charges on the external surface of the cell membrane,³⁶ displaces Ca²⁺ from binding sites on the surface of sarcolemmal membranes,³⁷ depletes sarcoplasmic reticulum Ca²⁺ stores, and after long exposures (>20 minutes) results in intracellular accumulation of La³⁺.³⁸ However, measurements of the external surface potential in the presence of different concentrations of La³⁺ (between 10^-7 to 10^-2 mol/L) indicate that the shift in the activation curve, which is also observed in our experiments, cannot result exclusively from the availability of this ion to screen and bind external negative surface charges.²⁶ The present results indicated that propafenone induced a concentration-dependent inhibition of I_K,s, being less potent in inhibiting I_K,s than in blocking I_K,r. On the other hand, the steady state inhibition of I_K,s did not depend on the voltage of the test pulse. Thus, the amount of block observed both on the I_Kmaxs and I_K,tails did not differ significantly for weak (-20-mV) or for strong (+70-mV) depolarizing pulses. As a consequence, at the two concentrations tested, propafenone (5 and 10 µmol/L) failed to produce any significant change either at the midpoint or in the slope of the activation curve of I_K,s. These results are similar to those reported with propafenone on I_K,r in rabbit atrial myocytes.¹² The lack of voltage-dependent effects on I_K,r and I_K,s differentiated propafenone from other class Ic antiarrhythmic agents, like flecainide and encainide, for which a strong correlation between voltage dependence of I_K,r activation and the observed degree of block has been demonstrated at low concentrations in cat ventricular myocytes.¹⁴

In the present study, the activation kinetics of I_K,s has been characterized by fitting the relation between tail amplitude and pulse duration to +30 mV to a monoexponential function of time after exclusion of the first data points to obtain the dominant time constant of activation. This procedure has been previously described and used for the same purpose³⁹ and has the advantage that it does not make any assumption about the gating mechanism of the slowly activating K⁺ channel. Under these conditions, 5 and 10 µmol/L propafenone did not significantly modify the time constant of the activation kinetics. Fig 5B shows that the ratio between the propafenone-sensitive current and the current in control conditions increased during the application of the depolarizing pulse, thus indicating that drug-induced block is linked to channel opening. In fact, propafenone-induced inhibition of other cardiac currents, including I_Na,⁸ I_to,¹³ and I_K,r,¹² is preferentially due to its interaction with the channels in their open state. Development of propafenone-induced block on I_K,s has been fitted by a monoexponential function. However, visual inspection of Fig 5B revealed that the single exponential fit misses some data points within the first second, thus suggesting an apparent lag in the development of block. Therefore, in an attempt to quantify the time course of development of block, the major dominant time constant was calculated from the exponential fits of these ratios after exclusion of the first data points. The results indicated that the time constant calculated by this procedure (which is the same used to calculate the time constant of the activation kinetics of the I_K,s) is fast (430 ms at +30 mV) and decreased at more depolarized levels, thus suggesting that block develops faster than current activation (time constant, 3.5 s). However, this observation does not exclude the possibility that block already starts during conformational states that appear during transitions between the rested and the open state,⁴⁰ as has been recently proposed to explain the I_K block induced by almokalant.³⁵ An analysis of unitary channel responses to the drug would help to more definitively identify underlying blocking mechanisms. An attempt to describe the time constant for block development by using pulses of different durations and measuring the amplitude of I_K,tails was also performed (see Fig 7). However, I_K,tails for pulses shorter than 0.7 s (when the block apparently develops on the maximum outward current) were very small under our experimental conditions (room temperature, depolarizing pulses to +30 mV), making reliable analysis difficult. Under these assumptions, since propafenone development of block is faster than the activation kinetics, it would be expected that I_K,s was simply scaled down without any noticeable effects on its activation kinetics. On the other hand, propafenone did not modify the time constant of deactivation of I_K,tails obtained on return to -30 mV after 5-s depolarizing pulses to +30 mV. A similar finding was observed by Duan et al¹² in rabbit atrial myocytes. It is expected that drugs that block slowly activating channels in the open state and exhibit a fast kinetics of recovery from block would induce a slowing on the deactivation kinetics of I_K,tails, since channels that become unblocked during the tail will generate outward current and, thus, slow the decay of the tail.^{14 35 41} The absence of an effect of propafenone on the time course of I_K,tail may suggest either that it binds to open channels but the drug exhibits a slow unblocking kinetics or that drug-bound channels can close during the repolarizing pulse. However, and since we have not characterized the deactivation kinetics at various potentials and after pulses to different voltages and durations, we cannot rule out any of these theoretical possibilities.

In other series of experiments, the possible use-dependent effects of propafenone on I_K,s were analyzed. In this group of experiments, the activation of K⁺ currents other than I_K,s was prevented by omitting the KCl and by adding La³⁺ and glibenclamide to the extracellular solution. Examination of the effects of propafenone on I_K,tails elicited during trains of pulses (Fig 9) revealed that a certain amount of block was apparent from the first applied depolarization (ie, tonic blockade⁴² ) and that with the repetition of the depolarizing pulse, the blockade slightly increased, particularly at fast (2-Hz) frequencies of stimulation. Thus, these observations would suggest that under our experimental conditions, propafenone-induced block is use dependent. It may further be stressed that in the present experiments no "reversed use dependence" was observed, since block of the first pulse was not relieved during a train of depolarizing pulses. The increase in the amount of block with successive depolarizations of the train was also evaluated by measuring the amplitude of the current elicited by each depolarization immediately after the capacitive transient. When this procedure was used, the "tonic blockade" was not observed, since the block developed within the first depolarization was avoided. Recently, it has been reported that dofetilide and E-4031 produced tonic and use-dependent block on I_K,r in rabbit ventricular myocytes when trains of pulses were applied.^{33 42} Thus, propafenone, dofetilide, and E-4031 are intermediate between two groups of drugs. One group is represented by quinidine, sotalol, disopyramide, tedisamil, and encainide, for which the block was developed from the first applied depolarization and did not significantly change with the repetition of the depolarizing pulse.⁴² The second group included drugs like amiodarone⁴² and almokalant,³⁵ for which the block appeared to be use dependent. The two types of propafenone-induced block on I_K,s observed during trains of pulses can be explained if it is considered that block develops so fast during depolarization that 50% of the total block induced by 5 µmol/L propafenone is attained during the first pulse. On the other hand, it has been described that the development of I_K,r block observed even at low concentrations of propafenone (0.1 µmol/L) is a fast process that was well fitted by a monoexponential function with a time constant of 476±77 ms.¹² According to these results, it could be speculated that within the first 200-ms depolarizing pulse of the trains, 34% of the propafenone-induced block on I_K,r would be reached ("tonic block") and, during the application of the train of pulses, the blockade would accumulate until a steady state was reached (use-dependent block). Therefore, and even when it would be of interest to analyze the possible use dependence of I_K,r block, it is possible that propafenone may produce similar effects on both components of I_K when trains of pulses were applied.

To further characterize the propafenone-induced block on I_K,s, the recovery process was analyzed by using a double-pulse protocol. In these experiments, currents elicited by the depolarizing test pulses were measured immediately after the capacitive transient. This procedure was used because 1-s depolarizing test pulses to +50 mV were long enough to ensure a complete development of block (time constant, 290 ms), and the effect of a change in interstimulus intervals on the amount of block is impossible to estimate. At very short interstimulus intervals, recovery from the propafenone-induced block apparently displays a delay. To characterize the dominant time constant of recovery, experimental data were adjusted to a monoexponential function after exclusion of these first data points. The results indicated that at depolarized levels (-40 mV), the recovery occurred with a time constant of 2.5 s. The combination of fast development of block during the depolarization and the lack of recovery during the short interstimulus intervals can explain the tonic and use-dependent block induced by propafenone on I_K,s when trains of pulses were applied. On the other hand, it will be necessary to analyze the recovery kinetics of the propafenone-induced block on I_K,r to better compare the effects of this drug on both components of I_K.

Recently, it has been demonstrated that the effects of propafenone on refractoriness were significantly increased at rapid rates, contributing to its ability to increase wavelength and terminate atrial fibrillation in dogs.⁴³ However, the relative importance of changes in APD and Na⁺ channel blockade in the effective refractory period changes caused by propafenone remains to be established. Moreover, propafenone blocks numerous channels; in fact, it has been described that in a narrow range of concentrations, it blocks all the currents that can play a role in the repolarization of cardiac action potential, ie, I_Na, I_Ca, I_to, I_K,r, I_K,s, and I_K1.^{8 9 12 13} Thus, its effects on action potential characteristics of different cardiac tissues (atrial and ventricular muscle or Purkinje fibers) and at different driving rates (at normal sinus rate or during a tachycardia) would be the result of its contradictory voltage- and time-dependent effects on each individual cardiac current. In addition, it should also be stressed that results in guinea pig ventricular myocytes (where both I_K,r and I_K,s are present) are quite different from those observed in other animal species (eg, cat or rabbit),^{16 33} where only I_K,r is present. Very recently, it has been demonstrated that I_K,s is present in a majority of human atrial cells, and the amplitude and kinetics of this current suggest that it is more likely to participate in phase 3 repolarization than is I_to.¹⁹ Unfortunately, the role of the slowly activating channels in human ventricular cells remains to be established. Therefore, analysis of the effects of antiarrhythmic drugs on I_K,s would lead to further insight into their possible effectiveness in the treatment of supraventricular tachyarrhythmias.

	Acknowledgments

 
This study was supported by CICYT SAF92-0157, Salud 2000, and Mapfre Foundation grants. The authors thank Dr L.M. Hondeghem and Dr D.J. Snyders for their valuable comments, Knoll Pharmaceuticals and Pfizer for providing the propafenone and dofetilide used in these studies, and Rubén Vara for his excellent technical assistance.

	Footnotes

 
Previously published as a preliminary report in abstract form (Biophys J. 1993;64:A314).

Received April 25, 1994; accepted November 1, 1994.

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