Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
0022-3565/99/2902-0515$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1999 by The American Society for Pharmacology and Experimental Therapeutics JPET 290:515–523, 1999 Vol. 290, No. 2 Printed in U.S.A. Mechanism of Transient Outward K1 Channel Block by Disopyramide JOSE A. SANCHEZ-CHAPULA Unidad de Investigacion “Carlos Mendez,” Centro de Investigaciones Biomedicas de la Universidad de Colima, Colima, Mexico Accepted for publication April 19, 1999 This paper is available online at http://www.jpet.org Potassium channels are critical for regulating excitability of cardiac myocytes, where they maintain the resting membrane potential, modulate action potential duration, and determine pacemaking activity. Potassium channels can be gated by voltage (Coraboeuf and Nargeot, 1993; Deal et al., 1996; Barry and Nerbonne, 1996), neurotransmitters such as acetylcholine, or intracellular ligands such as ATP, Ca21, or Na1 (Kurachi, 1995; Isomoto et al., 1997). Voltage-gated channels that activate and then inactivate very rapidly in response to membrane depolarization are called transient outward K1 channels. In the heart, these channels activate during the upstroke of the action potential and initiate the first phase of membrane repolarization. Rat ventricular myocytes are commonly used to study transient outward K1 current (Ito) because it is the major determinant of repolarization in these cells (Josephson et al., 1984). Class IA antiarrhythmic drugs block sodium channels and prolong action potential duration by blocking one or more K1 channels (Campbell, 1983). Examples of this class of antiarrhythmic agent are disopyramide and quinidine (Zipes and Troup, 1978). Microelectrode studies have shown that disopyramide depresses the maximum rate of repolarization, increases conduction time, and prolongs the terminal phase of Received for publication January 21, 1999. 1 This study was supported by Consejo Nacional de Ciencia y Tecnologia (Mexico) Grant 3729P-M. Ito at 270 mV was fast and best fit with a single exponential function having a time constant of 33 6 13 ms. In contrast, in the presence of 100 mM disopyramide, recovery from apparent inactivation was biexponential with time constants of 35 6 13 ms and 7.16 6 1.5 s. The time course of the slow component was used to estimate recovery of channels from block by disopyramide. Recovery from block was voltage-dependent, suggesting that disopyramide was trapped by the open channel. Taken together, these results suggest that disopyramide rapidly blocks channels in the open state and that unblock occurs from the inactivated state. cardiac repolarization (Kus and Sasyniuk, 1975). In isolated cardiac myocytes, disopyramide blocks sodium current in a use-dependent manner, probably by binding to the activated state of the channel (Sunami et al., 1991; Koumi et al., 1992; Zilberter et al., 1994). Disopyramide also blocks cardiac potassium currents, including the inward rectifier (Coraboeuf et al., 1988; Martin et al., 1994), the ATP-sensitive K1 current (De Lorenzi et al., 1995), the muscarinic receptor-operated K1 current (Watanabe et al., 1997), and Ito (Coraboeuf et al., 1988; Virag et al., 1997). In this study, we determined the mechanism of block of Ito channels by disopyramide in isolated rat ventricular myocytes. We conclude that disopyramide blocks the open state, and unblocks from the inactivated state of Ito channels. Materials and Methods Cell Preparation. Single ventricular myocytes were obtained from the right ventricular free wall of adult rats as described previously (Sanchez-Chapula, 1992). The hearts were mounted on a Langendorff apparatus and perfused for 5 min with normal Tyrode’s solution, then switched to a nominally calcium-free solution for an additional 5 min. Afterwards, the hearts were perfused for 20 min with a zero-calcium solution containing 1 mg/ml type I collagenase (Sigma, St. Louis, MO). The enzymes were washed out by perfusion with a high-potassium, low-chloride saline for 5 min. The free wall of the right ventricle was dissected away from the rest of the heart and cut into small pieces. Single cells were obtained by mechanical agi- ABBREVIATIONS: Ito, transient outward K1 current; IK, delayed rectifying outward K1 current; Isus, sustained current; 4-AP, 4-aminopyridine; HP, holding potential. 515 Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 ABSTRACT The block of the transient outward K1 current (Ito) by disopyramide was studied in isolated rat right ventricular myocytes using whole cell patch-clamp techniques. Disopyramide at a concentration of 10 to 1000 mM reduced peak Ito and accelerated the apparent rate of current inactivation. The onset of block was assessed using a double pulse protocol with steps from 270 to 150 mV. As the duration of the first (conditioning) pulse was increased from 1 to 50 ms, block was increased. Further prolongation of the conditioning pulse resulted in relief of block, which was nearly complete with a 1-s conditioning pulse. In the absence of drug, the recovery from inactivation of 516 Sanchez-Chapula Results Tonic Block of Ito by Disopyramide. Tonic effects of disopyramide on Ito were obtained in the presence of TEACa-Co external solution. Most of the experiments were performed in the TEA-containing solution unless otherwise indicated. Disopyramide decreased the peak outward current in a concentration-dependent manner, and accelerated the rate of apparent inactivation. Figure 1, A and B, shows Ito elicited by a voltage step from 270 to 150 mV in control, and in the presence of 100 and 300 mM. The concentration-response relationship for reduction of the integral of Ito by disopyramide is shown in Fig. 1C. The data was fit with a Hill equation to obtain a Kd of 259 mM and a Hill coefficient (nH) of 1.07. The time course of decay (inactivation) of Ito at 150 mV under control conditions was fitted by a single exponential function (Fig. 2C), with a time constant (t) 5 52 6 6 ms (n 5 14). In the presence of disopyramide, the time course of Ito decay was fitted with a biexponential function (Fig. 2B), with Fig. 1. Effects of disopyramide on Ito. A, superimposed currents traces of Ito obtained under control conditions and the presence of 100 mM disopyramide. B, effect of 300 mM disopyramide. C, concentration-response curve of the effect of disopyramide on the integral of Ito during a pulse of 200-ms duration. Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 tation with a pipette. The cells were maintained in a high-potassium, low-chloride solution at 4°C for up to 10 h before use in electrophysiological experiments. Solutions. Tyrode’s solution had the following composition (mM): 125 NaCl, 24 NaHCO3, 0.42 NaH2PO4, 5.4 KCl, 1.8 CaCl2, 1.0 MgCl2, 11 glucose, and 10 taurine. The solution was equilibrated with 95% O2/5% CO2, at pH 7.4. Nominally Ca- free solution was prepared by omitting CaCl2 from the Tyrode’s solution. The high-potassium, lowchloride solution had the following composition (mM): 80 K-glutamate, 50 KCl, 20 taurine, 3 KH2PO4, 10 glucose, 10 HEPES, and 0.2 EGTA. The pH was adjusted to 7.4 with KOH. The normal external solution had the following composition (mM): 140 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 11 glucose, pH adjusted to 7.4 by NaOH. The Ca-Co external solution had the following composition (mM): 140 NaCl, 4 KCl, 0.5 CaCl2, 2 CoCl2, 1 MgCl2, 10 HEPES, and 11 glucose; pH adjusted to 7.4 with NaOH. The TEA-Ca-Co solution had the following composition (mM): 90 NaCl, 50 TEA-Cl, 4 KCl, 0.5 CaCl2, 2 CoCl2, 1 MgCl2, 10 HEPES, and 11 glucose; pH adjusted to 7.4 with NaOH. All external solutions were equilibrated with O2 100%. Disopyramide (Sigma) was dissolved directly into the external solution to attain the desired final concentration. The internal (pipette) solution had the following composition (mM): 80 K-aspartate, 40 KCl, 10 KH2PO4, 1 MgSO4, 5 Na2ATP, 5 HEPES, and 5 EGTA. The pH was adjusted to 7.3 with KOH. Electrical Recording. A few drops of the cell suspension were placed in a chamber (0.5-ml volume) mounted on a modified stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). The chamber was superfused at a rate of 0.5 ml/min with normal external solution at room temperature (21–23°C). Currents were recorded using the whole-cell patch-clamp method (Hamill et al., 1981) and an Axopatch IC patch-clamp amplifier (Axon Instruments, Inc., Burlingame, CA). A Labmaster-TL/1 interface (Axon Instruments) controlled by pClamp 6.0 software (Axon Instruments) was used to generate voltage-clamp command protocols and acquire data. Currents were filtered at 2 kHz with a four-pole Bessel filter, digitally sampled at 4 kHz and stored on the hard disk of an Epson 486Dx/33 computer. Micropipettes were pulled from borosilicate glass capillary tubes (TW 150 – 6, World Precision Instruments, Sarasota, FL) on a programmable horizontal puller (Sutter Instruments, Novato, CA). When filled with the intracellular solution, the pipette tip resistance was 1 to 2 MV. Whole-cell capacitance and series resistance (Rs) compensations were optimized to minimize capacitive currents and reduce voltage errors. Protocols and Analysis. Cells with resting potentials of 275 mV or more negative were used for voltage clamp experiments. After membrane patch rupture, the cells were superfused with the Ca-Co or the TEA-Ca-Co external solutions. In rat ventricular myocytes, at least three different calcium-independent outward potassium currents activated by depolarization have been identified (Apkon and Nerbonne, 1991; Slawsky and Castle, 1994; Scamps, 1996). These include an Ito sensitive to 4-aminopyridine (4-AP), a delayed rectifying outward K1 current (IK) that activates and inactivates slowly and is sensitive to external TEA, and a sustained current (Isus) that is 4-AP- and TEA-insensitive. The goal of this study was to determine the mechanism of Ito block by disopyramide. Therefore, efforts were made to isolate Ito from other outward current components. TEA (50 mM) was used to block IK, a concentration that has no effect on Ito (Apkon and Nerbonne, 1991). Isus (Scamps, 1996) was measured from a holding potential (HP) of 210 mV to inactivate Ito (Apkon and Nerbonne, 1991; Slawsky and Castle, 1994). Isus measured with this protocol was digitally subtracted from currents elicited from a HP of 270 mV to obtain Ito (Slawsky and Castle, 1994). Data are expressed as mean 6 S.E.M. ANOVA with Student’s t test comparisons were used to compare the differences in mean values. A value of P , .05 was considered significant. Vol. 290 1999 Mechanism of Ito Block by Disopyramide 517 (1/k) of 279 mM, close to the Kd of 259 mM estimated from the concentration-response curve of Fig. 1C. Use-Dependent Effects of Disopyramide on Ito. Fig. 3, A and B, illustrates results obtained in an experiment designed to test for use-dependent block of Ito by disopyramide. A train of 16 pulses (30-ms duration, to 150 mV) was applied from an HP of 270 mV at a frequency of 1 Hz. Under control conditions, the current induced by each pulse remained constant during the pulse train. In the presence of 100 mM disopyramide, the amplitude of peak outward current during the first pulse was 15% less than control, and declined with successive pulses to reach a steady-state level equivalent to 60% of the control amplitude after four to six pulses. The average data from 12 cells is plotted in Fig. 3C. Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Fig. 2. A, inactivation time course of Ito under control conditions. Time course was fitted by one exponential with t 5 49.3 ms. B, inactivation time course Ito in the presence of 100 mM disopyramide. Time course was fitted by two exponentials with t1 5 16.9 ms and t2 5 174 ms. C, rate of block (1/tblock) as a function of the concentration. a fast time constant (tf) of 15 6 2 ms, and a slow time constant (ts) of 180 6 23 ms (n 5 14). The initial acceleration in the decay time course suggests that disopyramide could be an open channel blocker (Castle, 1990; Snyders et al., 1992; Clark et al., 1995). Further evidence that disopyramide is an open channel blocker is presented in Figs. 7 and 9. Therefore, tf for decay of current in the presence of disopyramide was assumed to approximate the rate of channel block (Snyders et al., 1992). Figure 2C shows the plot of 1/t (block) versus drug concentration for the data obtained from five experiments. The straight line is a least-squares linear fit to the relation: 1/ t ~ block! 5 kp @ D# 1 1 (1) The slope and intercept for the fitted relation yielded an apparent association rate (k) 5 0.19 * 106 M21 z s21 and dissociation rate (l) 5 53 s21. This yielded an apparent Kd Fig. 3. Use-dependent effects of disopyramide on Ito. A, superimposed records of pulses 1st, 2nd, 3rd, and 16th under control conditions. B, in the presence of 100 mM disopyramide. C, use-dependent development of block during the train of pulses. Peak Ito for each pulse in the train is normalized by dividing it by peak amplitude of Ito of the first pulse. Mean 6 S.D. of 12 cells is shown. Disopyramide produced a block during the first pulse of 21 6 5% and an additional use-dependent block of 26 6 6% with a t 5 0.68 pulse 21. 518 Sanchez-Chapula Recovery from Inactivation of Ito. Recovery of Ito from disopyramide-induced block was assessed with a paired voltage step protocol. A 100-ms conditioning pulse to 150 mV was used to inactivate Ito. The membrane potential was then clamped to 270 mV to allow recovery of channels from inactivation or from drug block. Recovery was assessed with a second pulse to 150 mV after a variable time at 270 mV. Recovery from inactivation of Ito was complete when the conditioning interval was .200 ms (Fig. 4A), and was best fit by a single exponential function with a time constant (tr) of 45.9 ms (n 5 16; Fig. 4C). In the presence of disopyramide, recovery was prolonged with a rapid and slow phase (Fig. 4B). The fast time constant was 51 6 11 and 49 6 013 ms in the presence of 30 and 100 mM disopyramide, respectively, similar to the single time constant of recovery (tr) found under control conditions. The slow time constant (ts) was two orders of magnitude greater than tr; 5211 6 822 ms at 30 mM, and 5048 6 755 ms at 100 mM disopyramide. The relative magnitude of the slow component of recovery was 0.22 6 0.08 at 30 mM and 0.35 6 0.11 at 100 mM disopyramide. Voltage-Dependent Onset and Recovery from Block. To determine if the use-dependent effects of disopyramide was voltage-dependent, the HP was varied between 240 and 290 mV during pulse trains to 150 mV. The duration of each pulse was 30 ms, and they were applied at a frequency of 1 Hz (Fig. 5A). The use-dependent block induced by 100 mM disopyramide was accentuated at more negative HPs. For example, the steady-state level of block was 7% at 240 mV and 47% at 290 mV. In Fig. 5B, the recovery from block in the presence of disopyramide 100 mM is shown at an HP of 250 and 290 mV. The onset of block was biexponential, with a tf of 85 ms at 250 mV and 26 ms at 290 mV. However, ts was 454 ms at 250 mV and 7900 ms at 290 mV. The values of tr for control, and tf and ts in the presence of disopyramide using an HP of 240 mV to 290 mV are shown in Fig. 5C. The value of tr and tf at all voltages was similar, suggesting that the fast component of recovery observed in the presence of disopyramide corresponds to the recovery from inactivation of drug-free Ito channels, and that ts reflects the recovery of channels from block. In contrast to tr and tf the values of ts increased at more negative membrane potentials. Effects of Duration of Conditioning Pulse on Recovery from Block. To determine if disopyramide blocks Ito channels in the inactivated state, recovery from block in the presence of drug was studied using conditioning pulses of 50 and 500 ms. Recovery from block was very slow after a 50-ms conditioning pulse (Fig. 6A), but when the duration of the conditioning pulse was increased to 500 ms, 95% of the peak amplitude was recovered after an interval of 150 ms (Fig. 6B). In Fig. 6C, the whole time course of the recovery from block is plotted for the two different conditioning pulses. The slow component of recovery had a similar time constant, 4870 6 675 and 5042 6 895 ms (n 5 7) with 50- and 500-ms conditioning pulses, respectively. However, the relative magnitude of the slow component of recovery was 0.46 6 0.06 after a 50-ms duration conditioning pulse, as opposed to 0.07 6 0.04 after a 500-ms conditioning pulse (n 5 7). These data show that block of Ito was reduced by prolonged depolarizations, suggesting unblock of drug from the inactivated state of the channel. Time Course of Block Onset by Disopyramide. The time course of disopyramide-induced block of Ito during a depolarizing pulse to 150 mV was determined using a paired pulse protocol from an HP of 270 mV. The duration of the first conditioning pulse was varied between 1 ms and 2 s. The duration of the second pulse was fixed at 100 ms. The conditioning and test pulses were separated by a gap of 300 ms at 270 mV to allow sufficient time for the channels that were not blocked by disopyramide during the conditioning pulse to recover from inactivation. Hence, comparisons of the test current amplitudes in the presence or absence of the conditioning pulse gave a measure of the disopyramide-induced block. Under control conditions, the amplitude of test current was only slightly (,5%) decreased when the duration of the con- Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Fig. 4. Disopyramide induced a component of slow recovery from block. We have used a standard two-pulse protocol to 150 mV (100-ms duration) from an HP of 270 mV. A, superimposed current traces under control conditions. B, current traces in the presence of 100 mM disopyramide. C, time course of the recovery from block under control conditions was complete in about 200 ms, fitted by a single exponential with a t 5 45 6 9 ms (mean 6 S.D.; n 5 6). In the presence of 30 and 100 mM disopyramide the process was biexponential, at 30 mM disopyramide the fast component time constant tf was 51 6 11 ms and slow component time constant ts was 5211 6 822 ms. In the presence of 100 mM drug tf was 49 6 13 ms and ts was 5048 6 755 ms. The magnitude of the slow component was 0.22 6 0.08 at 30 mM disopyramide and 0.35 6 0.11 at 100 mM. Vol. 290 1999 ditioning pulse was varied between 1 ms and 2 s (Fig. 7A). However, in the presence of 100 mM disopyramide, conditioning pulses as short as 3 ms produced a measurable decrease in test current amplitude, and pulses of longer duration resulted in greater depression of the test current. Maximal depression was reached with a 50-ms conditioning pulse. Further prolongation of the conditioning pulse increased the amplitude of the test current (Fig. 7B). In Fig. 7C, the normalized peak current amplitude was plotted as a function of the conditioning pulse duration. The decay phase (Fig. 7C, inset) was fitted by an exponential function with t 5 10.5 6 2.4 ms. The rising phase was fitted by a second exponential 519 Fig. 6. Recovery from block after 50 (A)- and 500 (B)-ms conditioning pulses, in the presence of 100 mM disopyramide. C, time course of recovery from block using both conditioning pulse durations. Time constants were similar using both protocols. However, the magnitude of the slow component was 46 6 6% using 50-ms duration conditioning pulse and 7 6 4% using 500-ms duration conditioning pulse (n 5 7 cells). with t 5 373 6 42 ms (n 5 6). To determine if the unblock induced by prolonged depolarization was related to the presence of TEA in the external solution, the same experiment was performed in the presence of a Ca-Co external solution (Fig. 8). Under control conditions, the increase in duration of the conditioning pulse produced a time-dependent depression of test peak current amplitude of 15 6 5% after a 2-s conditioning pulse (n 5 4). In the presence of Ca-Co external solution, the slowly inactivating IK current is present. IK exhibits a slow inactivation and recovery from inactivation behavior (t ; 500 ms; Apkon and Nerbonne, 1991), which can explain the depression of the current amplitude induced by increasing conditioning pulse duration. However, in the presence of disopyramide 100 mM, the unblocking effect induced by prolonged depolarization was still present. The effects of disopyramide on Ito in the presence of a Ca-Co external solution without TEA were similar to those obtained in the presence of TEA. The drug decreased peak current amplitude, accelerated the initial phase of the apparent inactiva- Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Fig. 5. A, modulation by HP of the use-dependent effects of disopyramide on Ito. Normalized peak Ito during trains of pulse in the presence of 100 mM disopyramide. The trains of 30-ms pulse to 150 mV at a frequency of 1 Hz were applied from different HPs. The use-dependent block was 7 6 3% at 240 mV, 31 6 9% at 260 mV, 39 6 8% at 270 mV, and 47 6 11% at 290 mV. B, time course of recovery from block of Ito in the presence of disopyramide, at different HPs. tf was 85 ms at 250 mV and 26 ms at 290 mV. ts was 454 at 250 mV and 7900 at 290 mV. C, voltage dependence of the recovery from inactivation (tr) under control conditions, and disopyramide tf and ts recovery from block. Mechanism of Ito Block by Disopyramide 520 Sanchez-Chapula Vol. 290 Fig. 7. Time course of disopyramide block during depolarization. Block was determined from the current during the test pulse to 150 mV applied after a conditioning pulse to 150 mV with variable duration (1–1000 ms) and a gap of 300 ms at 270 mV. A and B, selected tracings for conditioning pulses of 10-, 30-, 50-, 100-, 300-, and 500-ms duration. C, normalized peak current amplitude as a function of the duration of the conditioning depolarization. Note that block increased up to conditioning pulses of 50 ms, but declined with further prolongation of the conditioning depolarization. Inset shows the first 50 ms of the plot. The decaying phase was fitted by an exponential function with t 5 10.5 6 2.4 ms. The rising phase was fitted by another exponential function with t 5 373 6 26 ms (mean 6 S.D.; n 5 6 cells). tion, and slowed the last phase of the apparent inactivation (data not shown). These results suggest that the presence of TEA in the external solution did not modify the effects of disopyramide on Ito. Voltage Dependence of Depolarization-Induced Block by Disopyramide. The voltage dependence of activation of Ito and disopyramide-induced block was compared (Fig. 9). The voltage dependence of activation for Ito was measured from an HP of 270 by applying 15-ms pulses to potentials ranging from 230 and 1100 mV. After this activating pulse, the cell was immediately repolarized to 240 mV and the tail current amplitude after repolarization was used as a measure of Ito activation. Figure 9C (open circles) shows the voltage dependence of tail current amplitude, normalized to the peak at 160 mV. The membrane potential dependence of activation was fitted by a Boltzmann function, with the following equation: I/Imax 5 1/[1 1 exp(V m 2 V h/s)] (2) Vm is membrane potential, Vh is the voltage at which 50% of the channels are open, and s represents the slope factor for the relationship. Vh was 0.5 mV and s was 10.9 mV. The voltage dependence of the disopyramide-induced block of Ito was measured using a paired-pulse protocol (Fig. 9B). A conditioning pulse of 50-ms duration was applied to membrane potentials between 250 and 1100 mV from an HP of 270 mV. This pulse was followed by 300 ms at 270 mV and a test pulse to 150 mV. Under control conditions, this protocol produced less that 5% depression of Ito after the most positive conditioning potential. In the presence of 100 mM disopyramide, conditioning depolarizations positive to about Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Fig. 8. Time course of disopyramide block in the presence of a Ca-Co external solution. The protocol was the same as that used in Fig. 7. A, records obtained under control conditions. B, records obtained in the presence of 100 mM disopyramide. C, normalized peak current amplitude as a function of the duration of the conditioning pulse. Under control conditions, the peak current amplitude of the test pulse decreased as a function of the increase in duration of the conditioning pulse, reaching a 15 6 5% decrease after a 2-s duration conditioning pulse. In the presence of disopyramide block increased up to a conditioning pulse duration of 50 ms, but declined with further prolongation of the conditioning depolarization. 1999 Mechanism of Ito Block by Disopyramide 521 stant; and z, F, R, and T have the usual meanings. d represents the fraction of the transmembrane electrical field sensed by a single charge at the receptor site. The calculated values were 239 mM for Kd, and 0.19 for d (n 5 5). The Kd value obtained in these experiments is close to the Kd value obtained by different methods (Figs. 2 and 3B). It is clear that channel blockade has a steep voltage dependence coincident with channel opening and an additional weakly voltage-dependent component that reflects the effect of the transmembrane electrical field on the charged drug (Snyders et al., 1992). Discussion 240 mV produced measurable block of Ito and the amount of block increased at more positive potentials. The voltage dependence of disopyramide Ito block was plotted and fitted by the equation: y 5 $ 1/1 1 exp~~ V m 2 V h! /s% p $ D 1 K dp ~ 2z d FV/RT!% (3) where the first part of the equation is similar to the single Boltzmann function used to fit the activation curve. D is the disopyramide concentration; Kd is the apparent binding con- Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Fig. 9. A, current traces of an experiment studying voltage dependence of activation of Ito. Activation was measured by analysis of tail currents obtained after brief activating pulses (15 ms) to potentials between 230 and 100 mV. These activating pulses were followed by a test pulse to 240 mV. Tail currents were measured after pulses to 230, 210, 110, 120, 150, and 170 mV. B, current traces from the same experiment after superfusion with 100 mM disopyramide. From an HP of 270 mv, conditioning pulses of 50-ms duration to different voltages between 250 and 1100 mV were applied every 30s. These conditioning pulses were followed by a rest interval of 300 ms at 270 mV to allow the recovery from inactivation of Ito channels; the rest period was followed by the test pulse to 150 mV. Test pulses were preceded by conditioning pulses to 250, 0, 150, and 190 mV. C, steady-state voltage dependence of activation data were fitted by a Boltzmann function. The best fit was obtained with a Vh value of 0.5 mV and a slope factor of 10.9. The voltage dependence of block was fitted by eq. 3. The values for drug block (100 mM disopyramide) were Kd 5 239 mM and d 5 0.192. The main goal of the present work was to study the effect of disopyramide on Ito. Three different outward currents have been described in these cells (Apkon and Nerbonne, 1991; Slawsky and Castle, 1994; Scamps, 1996): 1) a rapidly activating and inactivating Ito, sensitive to 4-AP; 2) a more slowly activating and inactivating current IK, sensitive to external TEA; and 3) an apparently time-independent component Isus, insensitive to 4-AP and TEA. Ito was isolated from the two other types of outward currents by using external TEA (50 mM) to block IK and by digitally subtracting the noninactivating, time-independent Isus. Externally applied TEA has been shown to selectively block IK (Apkon and Nerbonne, 1991; Slawsky and Castle, 1994). The effects of disopyramide on Ito, including the decrease in peak amplitude, the initial acceleration of apparent inactivation, and the slowing of the late phase of repolarization were similar in the presence and in the absence of TEA. These findings suggest that TEA does not modify the tonic block of Ito by disopyramide. Disopyramide Is An Open Channel Blocker of Ito. The inhibition of Ito by disopyramide is characterized by a concentration-dependent reduction in peak Ito and an acceleration of the apparent rate of current inactivation. These results are similar to those found with different Ito open channel blockers like tedisamil (Dukes et al., 1990; Wettwer et al., 1998), bupivacaine (Castle, 1990), clofilium (Castle, 1991), quinidine (Slawsky and Castle, 1994; Clark et al., 1995), propafenone, and flecainide (Slawsky and Castle, 1994). The characteristics of the disopyramide-induced block of Ito strongly suggest that disopyramide blocks the open state of the channel. Evidence for this mechanism includes: 1) disopyramide accelerated the initial apparent inactivation rate of Ito, 2) at the onset of the depolarizing pulses there was no inhibition of Ito, indicating that disopyramide does not bind block channels in the rested state, 3) a close correlation between the voltage dependence of current activation and disopyramide-induced block, and 4) the unblock of Ito during prolonged depolarizations, which decrease the open state of the channel. Drugs that interact predominantly with the open state of the channel can do so by moving into the ion-conducting pore. If a positively charged drug moves into the membrane electrical field from the inside, the block should increase upon depolarization (Snyders et al., 1992). The data in Fig. 7 show that the voltage-dependent block induced by disopyramide consisted of two different phases. A very steep phase paralleled the voltage dependence of activation of the current (230 to 130 mV). The shallow phase probably reflects voltage 522 Sanchez-Chapula @ C# n 7 O 7 I 8 B where C, O, and I are closed, open, and inactivated states of the channel, respectively; n indicates that there is a series of several closed states leading to an open state (e.g., Zagotta and Aldrich, 1990); B is a nonconducting, disopyramideblocked channel. At positive membrane potentials, channel closure in the absence of drug occurs preferentially by inactivation. Hence, a prolonged depolarization results in rapid activation of the channel, followed by a slower decay. In the presence of disopyramide, drug block cannot occur until the channels open, and the decay of current will be initially accelerated by disopyramide because the open channel can close by two pathways, namely, inactivation (O 7 I) and disopyramide block (O 7 B). The model also includes a mutually exclusive interaction between drug block and channel inactivation, that is, blocked channels cannot inactivate and inactivated channels cannot be blocked. This constraint can explain the slowing of the final phase of apparent inactivation observed in the presence of disopyramide that produced a crossover of the current traces (Fig. 1; Wettwer et al., 1998). It can also explain the time-dependent development of block of Ito during the first 50 ms of the depolarizing pulses, whereas longer depolarizations induce unblocking of the channel (Fig. 8B). Comparison with Previous Studies. In rabbit ventricular myocytes, it was recently reported (Virag et al., 1998) that disopyramide decreased the amplitude of Ito by an open channel-blocking mechanism. However, this effect was not voltage- or use-dependent. In addition, disopyramide did not affect Ito recovery from inactivation. These results are in apparent contradiction to the results of the present work. Similar contradictory results have been obtained with quinidine. In rabbit atrial myocytes, Liu et al. (1996) found that quinidine induced a tonic decrease of Ito without additional use-dependent effects, but did not affect recovery from inactivation. However, in rat ventricular myocytes, quinidine produced significant tonic and use-dependent effects and a slowing of the Ito recovery from inactivation (Slawsky and Castle, 1994; Clark et al., 1995). Possible explanations for these apparent discrepancies are that the kinetics of recovery from inactivation of Ito in rabbit atrial and ventricular myocytes is a very slow process, with time constants in the range of seconds (Giles and Imaizumi, 1988), similar to the unblocking kinetics of disopyramide (this study). Another possibility is that the molecular bases of Ito and/or the Ito mechanism of block by disopyramide in rat are different than in rabbit. In rat ventricular muscle, it has been suggested that Kv 4.2 and Kv 4.3 isoforms contribute to Ito (see Fiset et al., 1997). The slow recovery from inactivation of Ito in rabbit cardiac myocytes suggests that Kv 1.4 could be the molecular basis of this current (see Yeola and Snyders, 1997). Disopyramide blocks other K1 currents, including IKr in rabbit ventricular myocytes (Carmeliet, 1993; Virag et al., 1998). Disopyramide has also been reported to block the inward rectifying potassium current IK1 and ATP-sensitive potassium current (Martin et al., 1994; De Lorenzi et al., 1995). These effects were voltage-dependent; the block increased steeply with depolarization and quickly decreased upon repolarization. As suggested in the present work, this profile of voltage dependence is consistent with a positively charged molecule blocking the channel from the intracellular side and entering the pore to such an extent as to be subjected to the transmembrane electrical field. Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 dependence block of the open channel. The fractional electrical distance defines the effect of the electrical field on the interaction between the drug and the receptor located in the channel. The value of (0.19) obtained for disopyramide indicates that the drug moves about 20% into the membrane electrical field to reach the receptor. This value for d is similar to that determined for quinidine block of Kv 1.5 (Snyders et al., 1992) and rat ventricular Ito (Clark et al., 1995). This similarity suggests that the structural determinants of the channel constrain binding to a particular location, such as the mouth of the inner vestibule. The slow recovery from block in the presence of disopyramide at the negative HPs (240 to 290 mV) can explain the use-dependent effect of the drug. We interpret the slow phase of recovery to represent the recovery of blocked channels during the interpulse interval. Recovery from block was slowed at more negative HPs. One possible explanation is trapping of the drug by the activation gate in the rested channel state. Because the chance to activate the channel is less at more negative membrane potentials, a slowing of recovery from block is expected. This phenomenon has been described for block of delayed rectifier K1 channels in squid giant axon by quaternary ammonium derivatives (Armstrong, 1971), and for some local anesthetics and antiarrhythmic drugs in neuronal and cardiac sodium channels (Yeh and Tanguy, 1985; Yeh and TenEick, 1987; Carmeliet, 1988). Competition between Drug Binding and Inactivation of Ito. We propose that disopyramide competes with the inactivation gate of the Ito channel. A key experiment that supports this proposal was performed in the presence of Ca-CoTEA external solution (Fig. 7). However, qualitatively similar results were found in a solution without TEA (Fig. 8). These results show that the presence of TEA in the external solution also did not modify the phasic effects of disopyramide on Ito. Therefore, we conclude that TEA did not modify the blocking effects of disopyramide on Ito. Prolonged membrane depolarization resulted in partial unblock of Ito channels. Evidence for this effect was obtained in three different types of experiments. First, in experiments studying recovery from block, increasing the duration of the conditioning pulse from 50 to 500 ms decreased the magnitude of the slow component of recovery without modifying the time constant. Second, experiments studying the time dependence of Ito block at 150 mV showed that the process was biphasic. During the first 50 ms there was an increase in block, but relief of block was observed with longer conditioning pulses. Third, when currents obtained by clamp pulses to 150 mV under control conditions and in the presence of disopyramide were superimposed, a crossover of the current traces was observed (Wettwer et al., 1998). These results suggest that disopyramide unbinds from the inactivated state of the channel. Moreover, our data suggest that drug binding and inactivation are mutually exclusive processes, that is, open-blocked channels do not inactivate, and inactivated channels are not blocked by the drug. Kinetic Scheme. To explain the disopyramide block of Ito, we propose a simple kinetic scheme: Vol. 290 1999 Acknowledgments We thank Dr. M. Sanguinetti for critical reading of the manuscript and editorial assistance, and Olivia Mercado Ruiz and Juan Carlos Muñoz for preparing the figures. References 523 tion process in guinea pig ventricular myocytes. J Pharmacol Exp Ther 261:1167– 1174. Kurachi Y (1995) G protein regulation of cardiac muscarinic potassium channel. Am J Physiol 269:C821–C830. Kus T and Sasyniuk BI (1975) The electrophysiological actions of disopyramide phosphate on canine ventricular muscle and Purkinje fibers. Circ Res 37:844 – 854. Liu L, Krinsky VI, Grant AO and Starmer F (1996) Cardiac transient outward potassium current: A pulse chemistry model of frequency-dependent properties. Am J Physiol 270:H386 –H397. Martin DK, Nakaya Y, Wyse KR and Campbell TJ (1994) Effects of disopyramide and flecainide on the kinetics of inward rectifier potassium channels in rabbit heart muscle. Br J Pharmacol 111:873– 879. Sanchez-Chapula J (1992) Caffeine inhibits depolarization-activated outward currents in rat ventricular myocytes. Eur J Pharmacol 229:163–169. Scamps F (1996) Characterization of a b-adrenergically inhibited K1 current in rat cardiac ventricular cells. J Physiol (Lond) 491:81–97. Slawsky MT and Castle NA (1994) K1 channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes. J Pharmacol Exp Ther 269:66 –74. Snyders DJ, Knoth KM, Roberds SL and Tamkun MM (1992) Time-, voltage- and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol Pharmacol 41:322–330. Sunami A, Fan Z, Nitta J-I and Hiraoka M (1991) Two components of use-dependent block of Na1 current by disopyramide and lidocaine in guinea pig ventricular myocytes. Circ Res 68:653– 661. Virag L, Varro A and Papp JG (1998) Effect of disopyramide on potassium currents in rabbit ventricular myocytes. Naunyn-Scmiedeberg’s Arch Pharmacol 357:268 – 275. Watanabe Y, Hara Y, Tamagawa M and Nakaya H (1997) Pirmenol inhibits muscarinic acetylcholine receptor-operated K1 current in the guinea-pig heart. Eur J Pharmacol 338:71–74. Wettwer E, Himmel HM, Amos GJ, Li Q, Metzger F and Ravens U (1998) Mechanism of block by tedisamil of transient outward current in human ventricular subepicardial myocytes. Br J Pharmacol 125:659 – 666. Yeh JZ and Tanguy J (1985) Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons. Biophys J 47:685– 694. Yeh JZ and TenEick RE (1987) Molecular and structural basis of resting and use-dependent block of sodium current defined using disopyramide analogues. Biophys J 51:123–135. Yeola SW and Snyders DJ (1997) Electrophysiological and pharmacological correspondence between Kv 4.2 current and rat cardiac transient outward current. Cardiovasc Res 33:540 –547. Zagotta WN and Aldrich RW (1990) Voltage-dependent gating of Shaker A-type potassium channel in Drosophila muscle. J Gen Physiol 95:29 – 60. Zilberter YI, Starmer CF and Grant AO (1994) Open Na1 channel blockade: Multiple rest states revealed by channel interactions with disopyramide and quinidine. Am J Physiol 35:H2007–H2017. Zipes DP and Troup PJ (1978) New antiarrhythmic agents: Amiodarone, aprindine, disopyramide, ethmozin, mexiletine, tocainide and verapamil. Am J Cardiol 41: 1005–1024. Send reprint requests to: Dr. Jose A. Sanchez-Chapula, CUIB, Universidad de Colima, Apdo. Postal 199, C.P. 28000, Colima, Col. Mexico. E-mail: [email protected] Downloaded from jpet.aspetjournals.org at ASPET Journals on May 2, 2017 Apkon M and Nerbonne JM (1991) Characterization of two distinct depolarizationactivated K1 currents in isolated adult rat ventricular myocytes. J Gen Physiol 97:973–1011. Armstrong CM (1971) Interaction of tetraethylammonium ion derivatives with the potassium channels of giant axons. J Gen Physiol 58:413– 437. Barry DM and Nerbonne JM (1996) Myocardial potassium channels: Electrophysiological and molecular diversity. Ann Rev Physiol 58:363–394. Campbell TJ (1983) Kinetics of onset of rate-dependent effects of class I antiarrhythmic drugs are important in determining their effects on refractoriness in guineapig ventricle, and provide a theoretical basis for their subclassification. Cardiovasc Res 17:344 –352. Carmeliet E (1988) Activation block and trapping of penticainide, a disopyramide analogue, in the Na1 channel of rabbit cardiac Purkinje fibers. Circ Res 63:50 – 60. Carmeliet E (1993) Use-dependent block of the delayed K1 current in rabbit ventricular myocytes. Cardiovasc Drugs Ther 7:599 – 604. Castle NA (1990) Bupivacaine inhibits the transient outward K1 current but not the inward rectifier in rat ventricular myocytes. J Pharmacol Exp Ther 255:1038 – 1046. Castle NA (1991) Selective inhibition of potassium currents in rat ventricle by clofilium and its tertiary homolog. J Pharmacol Exp Ther 257:342–350. Clark RB, Sanchez-Chapula J, Salinas-Estefanon E, Duff HJ and Giles WR (1995) Quinidine-induced open channel block of K1 current in rat ventricle. Br J Pharmacol 115:335–343. Coraboeuf E, Deroubaix E, Escande D and Coulombe A (1988) Comparative effects of three class I antiarrhythmic drugs on plateau and pacemaker currents of sheep cardiac Purkinje fibres. Cardiovasc Res 22:375–384. Coraboeuf E and Nargeot J (1993) Electrophysiology of human cardiac cells. Cardiovasc Res 27:1713–1725. De Lorenzi F, Bridal TR and Spinelli W (1995) Voltage-dependent inhibition of the ATP-sensitive K1 current by the class Ia agent disopyramide in cat ventricular myocytes. J Pharmacol Exp Ther 272:714 –723. Deal KK, England SK and Tamkun MM (1996) Molecular physiology of cardiac potassium channels. Physiol Rev 76:49 – 65. Dukes ID, Cleeman L and Morad M (1990) Tedisamil blocks the transient and delayed rectifier K1 currents in mammalian cardiac and glial cells. J Pharmacol Exp Ther 254:560 –569. Fiset C, Clark RB, Shimoni Y and Giles WR (1997) Shal-type channels contribute to the Ca21-independent transient outward K1 current in rat ventricle. J Physiol (Lond) 500:51– 64. Giles WR and Imaizumi Y (1988) Comparison of potassium currents in rabbit atrial and ventricular cells. J Physiol (Lond) 405:123–145. Hamill O, Marty A, Neher E, Sakmann B and Sigworth F (1981) Improved patchclamp techniques for high resolution current recording from cells and cell-free membrane patches. Pfluegers Arch 391:85–100. Isomoto S, Kondo C and Kurachi Y (1997) Inwardly rectifying potassium channels: Their molecular heterogeneity and function. JPN J Physiol 47:11–39. Josephson IR, Sanchez-Chapula J and Brown AM (1984) Early outward current in single rat ventricular cells. Circ Res 54:157–162. Koumi S-I, Sato R, Hisatome I, Hayakawa H, Okumura H and Katori R (1992) Disopyramide block of cardiac sodium current after removal of the fast inactiva- Mechanism of Ito Block by Disopyramide