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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
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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
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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.
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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.
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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.
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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.
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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-
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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.
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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-
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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
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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
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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-
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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
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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.
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Mechanism of Ito Block by Disopyramide