© 1999 American Heart Association, Inc.
Original Contributions |
Mechanism of Block and Identification of the Verapamil Binding Domain to HERG Potassium Channels
From the Section of Cardiology, University of Wisconsin, Madison, Wis.
Correspondence to Craig T. January, Section of Cardiology, Room H6/354 CSC, University of Wisconsin Hospitals and Clinics, 600 Highland Ave, Madison, WI 53792. E-mail ctj{at}medicine.wisc.edu
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Abstract—Calcium channel antagonists have diverse effects on cardiac electrophysiology. We studied the effects of verapamil, diltiazem, and nifedipine on HERG K+ channels that encode IKr in native heart cells. In our experiments, verapamil caused high-affinity block of HERG current (IC50=143.0 nmol/L), a value close to those reported for verapamil block of L-type Ca2+ channels, whereas diltiazem weakly blocked HERG current (IC50=17.3 µmol/L), and nifedipine did not block HERG current. Verapamil block of HERG channels was use and frequency dependent, and verapamil unbound from HERG channels at voltages near the normal cardiac cell resting potential or with drug washout. Block of HERG current by verapamil was reduced by lowering pHO, which decreases the proportion of drug in the membrane-permeable neutral form. N-methyl-verapamil, a membrane-impermeable, permanently charged verapamil analogue, blocked HERG channels only when applied intracellularly. Verapamil antagonized dofetilide block of HERG channels, which suggests that they may share a common binding site. The C-type inactivation-deficient mutations, Ser620Thr and Ser631Ala, reduced verapamil block, which is consistent with a role for C-type inactivation in high-affinity drug block, although the Ser620Thr mutation decreased verapamil block 20-fold more than the Ser631Ala mutation. Our findings suggest that verapamil enters the cell membrane in the neutral form to act at a site within the pore accessible from the intracellular side of the cell membrane, possibly involving the serine at position 620. Thus, verapamil shares high-affinity HERG channel blocking properties with other class III antiarrhythmic drugs, and this may contribute to its antiarrhythmic mechanism.
Key Words: Ca2+ antagonist • HERG • rapidly activating delayed K+ channel • antiarrhythmic drug • arrhythmia
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L-type Ca2+ channel antagonists (phenylalkylamines, benzothiazepines, and dihydropyridines, with respective prototype drugs verapamil, diltiazem, and nifedipine) have distinct effects on cardiac electrophysiology and arrhythmias.1 In heart cells, low concentrations of verapamil have been shown to prolong cardiac action potential duration, whereas high concentrations shorten it.2 3 4 In contrast, dihydropyridines cause only concentration-dependent shortening of cardiac action potential duration.4 5 Some forms of ventricular tachycardia are specifically sensitive to verapamil.6 7 Verapamil has recently been shown to prolong the atrial effective refractory period in humans and to prevent atrial tachycardia- and fibrillation-induced shortening of the atrial effective refractory period ("electrical remodeling").8 9 Observations such as these suggest that in addition to blocking L-type Ca2+ channels, verapamil may modify repolarizing membrane currents at low drug concentrations.
Cardiac delayed rectifier K+ current is composed of 2 distinct currents, the rapidly (IKr) and slowly (IKs) activating components.10 IKr channel protein is encoded by the human ether-a go-go-related gene (HERG).11 12 13 Suppression of HERG channels causes action potential and QT interval prolongation, which can be both antiarrhythmic and cause long-QT syndrome.14 We examined the effects of verapamil, diltiazem, and nifedipine on HERG channels heterologously expressed in a human embryonic kidney (HEK 293) cell line. Our data show that verapamil is a potent antagonist of HERG channel current, whereas diltiazem only weakly suppresses HERG current, and nifedipine has no effect. We then studied verapamil block of HERG channels, including its use- and frequency-dependent properties, membrane sidedness of action, and binding domain on the HERG channel. Our results indicate that externally applied verapamil enters the membrane in the neutral form to reach a binding domain within the pore that is accessible from the intracellular side of the membrane. Preliminary reports of this work have appeared.15 16 17
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DNA Constructs and Stable Transfection of HEK 293 Cells
The transfection techniques have been published previously.13 Briefly, HERG cDNA was subcloned into BamHI/EcoRI sites of the pCDNA3 vector (Invitrogen). HEK 293 cells were transfected with this construct using the lipofectamine method (GIBCO), and the stably transfected cells were subcloned to achieve a uniform HERG expression level. cDNA for the HERG mutations Ser631Ala and Ser620Thr (provided by Dr Gail Robertson, University of Wisconsin, Madison) was expressed transiently or stably in a similar manner. The cells were cultured in MEM supplemented with 10% FBS and 400 µg/mL G418. The cells were harvested from the culture dish by trypsinization, washed twice with standard MEM, and stored in this medium at room temperature for electrophysiological study.
Patch-Clamp Recording Method
Aliquots of cells were allowed to settle on the bottom of a
<0.5 mL cell bath mounted on an inverted microscope (Diaphot, Nikon).
Cells were superfused with Tyrode solution containing (in mmol/L)
137 NaCl, 4 KCl, 1.8 CaCl2, 1
MgCl2, 10 glucose, and 10 HEPES (normal pH 7.4
adjusted with NaOH). Solution exchanges in the cell bath were completed
within 1 minute. Membrane currents were recorded in the whole-cell
patch clamp configuration. The pipette had inner diameters of 1 to
1.5 µm and had resistances of 2 to 4 M
when filled with
the internal pipette solution. The internal pipette solution contained
(in mmol/L) 130 KCl, 1 MgCl2, 5 EGTA, 5
MgATP, and 10 HEPES (pH 7.2 adjusted with KOH). A Dagan 3911A patch
clamp amplifier was used to record membrane currents. Computer
software (pCLAMP6, Axon Instruments) was used to generate voltage clamp
protocols, acquire data, and analyze current signals. All
experiments were performed at room temperature (23±1°C).
Drugs and Chemicals
Verapamil, diltiazem, and nifedipine
were purchased from Sigma. Verapamil and diltiazem were
dissolved in distilled water to make 1 mmol/L and 10 mmol/L
stock solutions, respectively. Nifedipine was dissolved in
DMSO to give a stock concentration of 50 mmol/L.
N-methyl-verapamil, a quaternary, permanently
charged, membrane-impermeable verapamil analogue, was
obtained from Dr G. Paul (Knoll Pharmaceuticals, Ludwigshafen, Germany)
as N-methyl-verapamil hydrochloride and was
dissolved in distilled water to make a 10 mmol/L stock solution.
Dofetilide
{N-[4-(-{-[4-(methanesulfonamino-phenoxyl]-N-methylethylamino}ethyl)phenyl]
-methanesulfonamide} was obtained from Pfizer Central Research and
was dissolved in acidic water to make a 100 µmol/L stock
solution. Final drug concentrations were made by diluting stock
solution with Tyrode or pipette solution. DMSO at a concentration
(0.1%) equivalent to the highest drug dilution studied had no effect
on HERG current (n=4 cells).
Curve Fitting and Statistical Methods
Data are given as mean±SEM. Curve fitting was done using
multiple nonlinear least-squares regression analysis (Origin,
Microcal Software; Clampfit, Axon Instruments). Concentration effects
were quantified by fitting the Hill equation
{Idrug/Icontrol=1/[1+ (D/IC50)n],
where D is the drug concentration,
IC50 is the drug concentration for 50% block,
and n is the Hill coefficient} to the results. Statistical
significance was analyzed using a Student t test or
ANOVA, where appropriate. P<0.05 was considered
statistically significant.
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Verapamil, Diltiazem, and Nifedipine Effects on HERG Channels
Figure 1A
shows control HERG current
elicited by depolarizing steps to between –70 and 60 mV for 4 seconds
applied from a holding potential of –80 mV. Tail current was
recorded after the repolarizing step to –50 mV. Figure 1B
shows the effect of 2 µmol/L verapamil on HERG
current in the same cell. Figure 1C shows averaged
current-voltage (I-V) relations for HERG current measured at
the end of depolarizing steps and for the subsequent tail current peak
amplitude for control conditions and with 2 µmol/L
verapamil in the same cells (n=4 cells). Under control
conditions, HERG current activated at voltages positive to –50
mV, maximum current was reached for steps to –10 mV, and at more
positive voltages inward rectification was present because of
voltage-dependent rapid inactivation.18 19 The tail
current was maximal after voltage steps positive to 0 mV. At a
concentration of 2 µmol/L, verapamil block of HERG
current was nearly complete at all voltages. Figures 1D and 1E
show the reversibility of verapamil block. In panel D, HERG
current was activated with a step to 20 mV, and tail current
was recorded after the step to –50 mV. The voltage protocol was
repeated at 15-second intervals, and HERG tail current peak amplitude
plotted versus time is shown in panel E. Exposure to
verapamil (500 nmol/L) caused a rapid reduction in current
amplitude to a steady level of block that was rapidly reversed after
drug washout. To study the concentration dependence relation for block
of HERG current by verapamil, 4 to 9 cells were studied at
each drug concentration, with each cell exposed to only 1 drug
concentration. Verapamil block of HERG current at the end
of the depolarizing step to 20 mV and the tail current peak amplitude
were normalized to the respective control values and are plotted as
relative current amplitude in panel F. Data points were fit with the
Hill equation. The half-maximal inhibition concentration
(IC50) for block of tail current was 143.0 nmol/L
with a Hill coefficient of 1.06, whereas the IC50
for block of current measured at the end of the depolarizing step to 20
mV was 160.0 nmol/L with a Hill coefficient of 0.91. As shown in panel
F, these concentration dependence relations were nearly identical. The
Hill coefficients are consistent with the binding of
verapamil to a single site.
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and 
) and tail current peak amplitude
(Itail, 
and 
) in the absence (
The concentration dependence for block of HERG current by diltiazem was
studied in 5 to 8 cells at each drug concentration using a protocol
similar to that shown in Figure 1D
. Diltiazem blocked HERG
current in a concentration-dependent manner, and diltiazem block of
HERG tail current peak amplitude is shown in Figure 1F. Data
were fit with the Hill equation to give an IC50
of 17.3 µmol/L that is 100 times higher than that of
verapamil. The Hill coefficient was 1.03,
consistent with the binding of drug to a single site. In
contrast to verapamil (see below), diltiazem block of HERG
current was present immediately on depolarization, and no recovery
of HERG current was observed after repolarization to –80 mV for up to
10 minutes; diltiazem block, however, was reversible with drug washout.
The effect of nifedipine on HERG current was studied at
concentrations up to 50 µmol/L using a protocol similar to that
shown in Figure 1D. Despite the use of repetitive pulses and
drug exposure periods of up to 30 minutes, nifedipine had
no effect on HERG current amplitude (n=6 cells). These data are plotted
in Figure 1F. A similar lack of effect of nifedipine
was observed when the holding potential was –50 mV (data not shown),
which accentuates dihydropyridine block of L-type
Ca2+ channels.20
Intracellularly, but Not Extracellularly, Applied
N-Methyl-Verapamil Blocks HERG
Channels
To test whether verapamil acts on HERG channels from
the outside or inside of the cell membrane, we studied the effects of
verapamil and N-methyl-verapamil, a
permanently charged, membrane-impermeable analogue. HERG current was
activated from a holding potential of –80 mV by a step to 20
mV for 4 seconds, followed by a step to –50 mV for 6 seconds to
record tail current, with the protocol repeated at 15-second
intervals. Figure 2A shows original
current traces (left panel) and tail current peak amplitude plotted as
a function of time (right panel). The extracellular application of
1 µmol/L verapamil reduced HERG tail current to
9.9±5.2% of the control value (n=6 cells, P<0.01), which
was reversed with drug washout. Figure 2B shows original current
traces (left panel) and tail current peak amplitude plotted versus time
(right panel) with the addition to the bath of 20 µmol/L
N-methyl-verapamil. It had virtually no effect
on HERG tail current peak amplitude, which was 91.4±3.6% of the
control value after 20 minutes of exposure (n=4 cells,
P>0.05). Thus, the extracellular application of the
membrane-impermeable N-methyl-verapamil analogue
did not block HERG channels.
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We then tested its effect by intracellular application.
N-methyl-verapamil (2 µmol/L) was
included in the pipette solution and immediately after obtaining
whole-cell clamp HERG current was recorded using the same
voltage protocol. Figure 2C shows original current traces (left
panel) and tail current peak amplitude plotted versus time (right
panel). HERG tail current peak amplitude decreased with time as
N-methyl-verapamil diffused from the pipette
into the cell, and this could be fit as a single-exponential decay with
an average time constant of 2.3 minutes. By 10 minutes of whole-cell
recording, the current had decreased to 35.8±1.9% (n=4 cells,
P<0.05) of the initial value. In control experiments
without N-methyl-verapamil in the pipette, HERG
tail current peak amplitude decreased to 96.2±3.1% (n=4 cells,
P>0.05) of the control value over the same time interval,
which confirms that the decrease in HERG current was due to internally
applied N-methyl-verapamil.
Effects of pHo on Block of HERG Channels by
Verapamil
Verapamil is a weak base, and the fractional
distribution of drug between neutral (membrane permeable) and
positively charged (membrane impermeable) forms can be controlled by
varying pH. We used this to evaluate further whether
verapamil block of HERG channels required the neutral form
of the drug. This approach has been previously used to help to
elucidate the membrane sidedness of drug action on ion
channels.21 22 The dissociation constant (pKa) of
verapamil in solution has been estimated to be between 8.73
and 8.99.23 Assuming a pKa of 8.8, at pH 8.4, 28% of
verapamil would be in the neutral form. As shown in Figure 3A, at this pHO the
extracellular application of 500 nmol/L verapamil reduced
HERG tail current peak amplitude to 17.0±3.4% of the control value
(n=4 cells). At a pHO of 7.4, at which 4.0%
of the drug is in the neutral form, 500 nmol/L verapamil
reduced HERG tail current to 22.4±2.4% (n=9 cells), and at
pHO 6.4, at which 0.4% of
verapamil is in the neutral form, 500 nmol/L
verapamil reduced HERG tail current only to 81.9±3.2%
(n=6 cells) of the control values. Thus, changes in
pHO that decrease the amount of drug in the
neutral, membrane-permeable, form decreased drug block of HERG
channels, which suggests that the neutral form of verapamil
is required for HERG channel block. Figure 3A also shows that
changing pHO for control conditions had
additional effects on HERG current. Particularly changing
pHO from 7.4 to 6.4 decreased HERG current
amplitude and markedly accelerated the HERG tail current decay. The
effects of pHO on HERG current require further
investigation and will be reported separately.
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We studied the effect of pHO on block by
N-methyl-verapamil, to confirm that changing
pHO did not alter the extent of block when the
permanently charged analogue was applied intracellularly. As shown in
Figure 3B, when applied intracellularly through the pipette,
2 µmol/L N-methyl-verapamil blocked HERG
current. At a pHO of 7.4, HERG tail current peak
amplitude was reduced to 33.0±1.2% of the initial value, and at a
pHO of 6.4, HERG tail current peak amplitude was
reduced to 27.0±3.2% of the initial value (n=4 cells,
P>0.05). As expected, altering pHO
did not affect block by N-methyl-verapamil. The
results shown in Figures 2 and 3 suggest that
verapamil permeates the cell membrane in a neutral form and
that its binding site to the HERG channel is accessible from the
internal side of the cell membrane.
Development of Drug Block by Verapamil
HERG block by verapamil was use and frequency
dependent, as shown in Figure 4. In
Figure 4A, 200 nmol/L verapamil, a concentration
close to the IC50, was washed in for 10 minutes
while the cell was held continuously at –80 mV to maintain HERG
channels in a closed state. HERG channels were then rapidly
activated by a depolarizing step to 60 mV for 200 milliseconds
and then to –50 mV for 200 milliseconds to elicit tail current, before
repolarizing to the holding potential. Trains of these pulses were
applied at intervals of 0.6, 2, or 20 seconds, with each cell studied
only at 1 pulse frequency, and the development of drug block was
plotted versus time. The time course of the development of HERG block
was dependent on the frequency of HERG channel activation, which
suggests activated (open or inactivated) state
block by verapamil. At each pulse frequency, 10 to 15
pulses were required for a steady level of block to be reached. The
extent of drug block was weaker at lower pulse frequencies, which
suggests time-dependent drug unbinding at the holding potential between
pulses.
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We used a second voltage protocol to characterize further properties of
verapamil block of activated channels, as shown in
Figure 4B. From the holding potential, HERG channels were
rapidly activated by a 100-millisecond step to 60 mV, which was
followed by a 10-second step to 0 mV. Tail current was then elicited
with a 6-second step to –50 mV. For control conditions, at 0 mV HERG
current amplitude was nearly constant, and the subsequent repolarizing
step to –50 mV caused a tail current that decayed gradually. The cell
was then held continuously at –80 mV for 10 minutes to maintain
channels in a closed state during verapamil (500 nmol/L)
washin. With the first depolarization in the presence of
verapamil, the initial HERG current amplitude was unchanged
compared with the control current, indicating minimal rested (closed)
state block. The current amplitude then declined during the maintained
depolarization at 0 mV to reach a steady level of drug block. In 9
cells studied with this protocol, the current remaining at the end of
the 10-second step to 0 mV declined to 28.6±2.3% of the initial
value, and when fit as a single exponential it decayed with a time
constant of 0.93±0.08 seconds. Tail current peak amplitude was
similarly reduced to 22.4±2.4% of the control value. More
importantly, the tail current recorded with verapamil
exposure was slower to decay and it "crossed over" (Figure 4B, arrow) the control tail current trace to become larger in
amplitude. When the step to –50 mV was increased to 60 seconds
duration, the tail currents decayed to the same value (data not shown).
These effects of verapamil were reversed with drug
washout.
We studied inactivation properties of HERG current in control
conditions and with verapamil present at a
concentration (200 nmol/L) near the IC50. In 1
series of experiments, cells were held at 60 mV and hyperpolarized to
–100 mV for 10 milliseconds, which is sufficient at room temperature
to remove inactivation and maximize occupancy in the open state. A test
step to 0 or 60 mV was then applied to elicit a large-amplitude outward
current that rapidly decayed as HERG channels
inactivated.13 18 19 The rate of inactivation
was measured for each cell by fitting a monoexponential
function to the decaying outward current. At test potentials of 0 and
60 mV, time constants in the absence of verapamil were
8.9±0.6 and 3.0±0.3 milliseconds (n=9 cells), respectively. With 200
nmol/L verapamil in the extracellular perfusate,
the time constants were 8.7±0.6 and 2.9±0.3 milliseconds (n=9 cells),
respectively, which are not different from the control values
(P>0.05). We also studied the effects of
verapamil on recovery from inactivation. The cells were
depolarized to 60 mV for 200 milliseconds to rapidly
inactivate HERG channels, and the cells were then
repolarized to –50 mV to elicit a tail current. The rising phase
("hook") of the tail current represents the rapid recovery
of HERG channels from inactivated to open states, whereas
the slow decay phase of tail current represents HERG channel
deactivation.13 18 19 After performing a
double-exponential fit to the tail current trace, the time constant for
the rising phase was used to measure recovery from inactivation. At
–50 mV, the time constant of recovery from inactivation was 7.3±0.3
milliseconds, and in the presence of 200 nmol/L verapamil
it was 7.2±0.3 milliseconds (n=18 cells, P>0.05). Thus,
verapamil exposure did not affect the apparent rates of
inactivation and of recovery from inactivation of HERG channels. The
rate of deactivation, as shown in Figure 4B, was markedly
slowed.
Verapamil and
N-Methyl-Verapamil Unbind From HERG Channels
During Repolarization
HERG channels recover from verapamil or
N-methyl-verapamil block on repolarization
despite continued drug exposure. As shown in Figure 5A, a 100-millisecond step was applied to
60 mV to rapidly activate HERG current and was followed by a
10-second conditioning step to 0 mV to obtain a steady level of drug
block by verapamil (500 nmol/L). The membrane was then
repolarized for a variable time to –50, –80, or –110 mV. A test
pulse (step to 60 mV for 100 milliseconds and then to 0 mV for 200
milliseconds) was then applied to assess the recovery of HERG current
from drug block. The peak amplitude of HERG current during the test
pulse at 0 mV was normalized to that during the conditioning step at 0
mV in 3 to 6 cells studied at each recovery voltage, and peak
amplitudes are plotted as a function of the recovery time (
time) in
Figure 5B. At the onset of the recovery interval, the relative
current was 25%, which represents the steady-state current
remaining at the end of the preceding conditioning step (see Figure 5A). HERG current amplitude elicited with the test steps
increased with longer recovery times as verapamil unbound
from channels, and by 10 minutes of recovery 80% of HERG current
could be elicited. The fraction of current that recovered was fit with
2 exponentials. At each recovery voltage, recovery from drug block
contained a rapid component with similar time constants (
1)
of 0.9 to 1.1 seconds and amplitudes (A1) of 0.18 to 0.24. The
slow-component time constant (2) showed prominent voltage
dependence, increasing at more negative recovery voltages from 52 to
285 seconds with amplitudes (A2) of 0.28 to 0.39.
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Data for the permanently charged
N-methyl-verapamil (2 µmol/L in the
pipette solution) analogue were obtained with the same voltage protocol
and are shown in Figure 5C. Three to six cells were studied at
each recovery voltage. Recovery of HERG current at –50 mV was fit by
rapid (1 of 3.5 seconds) and slow (2 of 73.5 seconds) time
constants with amplitudes of 0.19 and 0.43, respectively. At recovery
voltages of –80 or –110 mV, the fitting procedure generated rapid
time constants with very small amplitudes (A1 of 0.03), and recovery of
HERG current occurred almost entirely through the slow time constant
(A2 of 0.59 and 0.43, respectively). The slow-component time constant
(2) also showed prominent voltage dependence, increasing at more
negative recovery voltages from 74 to 338 seconds.
Antagonism of Dofetilide Block of HERG Channels by
Verapamil
The methanesulfonanilide antiarrhythmic drug dofetilide blocks
IKr and HERG channels in nanomolar
concentrations, and it is thought to bind to the internal pore of the
channel at a site that involves the serine residue at position
620.24 If verapamil and dofetilide
compete for a common receptor site, then binding to the receptor by one
drug should interfere with binding of the other drug. We tested whether
verapamil could antagonize dofetilide binding. In these
experiments, HERG channels were activated from the holding
potential of –80 mV by a depolarizing step to 20 mV for 4 seconds,
which was followed by a repolarizing step to –50 mV for 6 seconds to
elicit tail current, and the protocol was repeated every 15 seconds.
HERG tail current peak amplitude (normalized to the initial value) was
plotted versus time, and the period of drug application is indicated by
the horizontal line(s). In Figure 6A, the
application of 10 µmol/L verapamil resulted in the
complete block of HERG current. With 15 minutes of washout the tail
current amplitude recovered to 89.6±2.4% (n=4 cells; see Figure 6D) of control. In Figure 6B, the application of 100
nmol/L dofetilide also resulted in the complete block of HERG current.
In contrast to verapamil, recovery of HERG current after
dofetilide washout was minimal, and at 15 minutes of washout only
7.4±2.9% (n=5 cells; see Figure 6D) of tail current amplitude
had recovered. We then tested for antagonism of dofetilide binding by
verapamil. As shown in Figure 6C, HERG current was
first blocked by 10 µmol/L verapamil, and 100 nmol/L
dofetilide was applied, as in Figure 6B. Dofetilide was then
washed from the chamber, followed 2.5 minutes later by
verapamil with the gradual recovery of HERG current. With
15 minutes of washout, the HERG tail current amplitude recovered to
51.9±7.8% (n=5 cells; see Figure 6D) of control
(P<0.01 compared with dofetilide alone). Thus, pretreatment
with verapamil prevented dofetilide binding and permitted
the recovery of HERG current with drug washout. These results suggest
that verapamil and dofetilide may compete for a common
receptor site, although an allosteric interaction between separate
verapamil and dofetilide binding sites cannot be
excluded.
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C-Type Inactivation-Deficient Mutations Modify
Verapamil Block
To study further verapamil block of HERG channels, 2
HERG mutations, Ser620Thr and Ser631Ala, were evaluated. Previous
studies have shown that these mutations interfere with C-type
inactivation and modify block of methanesulfonanilide antiarrhythmic
drugs of HERG channels.24 25 26 The Ser620Thr mutation lies
in the S5-S6 linker near the internal mouth of the channel pore, and
the Ser620 residue has been identified as critical to the binding of
dofetilide to the channel.24 The Ser631Ala mutation lies
near the external mouth of the channel pore. In our experiments, both
mutations disrupt HERG current inactivation (see Figure 7A and 7B). Verapamil, at a
concentration that abolishes wild-type HERG current (10 µmol/L),
only weakly blocked current present with the Ser620Thr mutation,
whereas it blocked nearly completely current present with the
Ser631Ala mutation. The concentration dependence relations for
verapamil block of wild-type (see Figure 1F),
Ser620Thr, and Ser631Ala channel currents are shown in Figure 7C. For the Ser620Thr mutation, the IC50
for verapamil block of HERG current at the end of the
depolarizing step to 20 mV was increased 444-fold to 71.1 µmol/L
(n=7 cells) from 0.16 µmol/L for wild-type HERG channels. In
contrast, the IC50 for verapamil
block of the Ser631Ala mutation increased 22-fold to 3.45 µmol/L
(n=7 cells) compared with that for wild-type HERG channels.
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Calcium Channel Antagonist Effects on Cardiac Ion Channels
Calcium channel antagonists block L-type Ca2+ channels with use- and voltage-dependent properties, and wide ranges of values have been reported for the concentration dependence of block.20 27 In general, phenylalkylamines block native and cloned L-type Ca2+ channels with IC50 values in a range of 250 nmol/L to at least 15.5 µmol/L. Benzothiazipines produce block in a similar concentration range, whereas dihydropyridines can produce more potent Ca2+ channel block. Our experiments with HERG K+ channels showed a different drug sensitivity pattern than that found for block of L-type Ca2+ channels and suggest that verapamil blocks of HERG K+ channels in a concentration range similar to that required for block of L-type Ca2+ channels.
Calcium channel antagonists are also known to block several K+ channels; however, block generally has required relatively high drug concentrations. For example, methoxy-verapamil (D600) at concentrations of 2 to 10 µmol/L suppressed delayed rectifier K+ current (IK) in calf Purkinje fibers5 and cat ventricular cells,28 whereas in frog atrial cells IK was blocked by D600 but only at very high concentrations (IC50=820 µmol/L).29 Verapamil blocked hKv1.5 K+ channels expressed in HEK 293 cells with an estimated KD of 21.1 µmol/L.30 Limited data are available for IKr and HERG channels. In guinea pig ventricular myocytes, verapamil (1 µmol/L) was recently reported to suppress IKr tail current by 49%,4 and in transfected COS cells some Ca2+ channel antagonists, including verapamil (EC50=0.83 µmol/L), recently have been reported to suppress HERG current,31 although in these reports the mechanism of drug block was not examined. Our findings agree with these recent reports, although the IC50 that we found for verapamil block of HERG tail current (143 nmol/L) is slightly lower, and diltiazem in higher drug concentrations completely blocked HERG channels in our experiments. These results provide a cellular mechanism for previous reports of action potential prolongation with low verapamil concentrations.2 3 4
Characteristics of HERG Channel Block and Unblock by
Verapamil
Our experiments showed that block of HERG channels by
verapamil required channel activation, as drug binding did
not occur to the rested, or closed, state. Verapamil also
unbinds from HERG channels on repolarization to voltages close to the
normal cardiac cell resting potential. As shown in Figure 4B, the tail current decay at –50 mV in verapamil was slower
and crossed over the control tail current decay. A similar crossover
occurred with N-methyl-verapamil suppression of
HERG current (see Figure 2C). The crossover can be explained by
time-dependent verapamil unbinding from HERG channels, with
the drug unbound channels then opening before deactivating. Although
the crossover has been ascribed to drug unbinding from open
channels,32 drug unbinding from
inactivated HERG channels that then recover through the
open state also could cause a crossover. Our results do not directly
distinguish between these possibilities. The finding that the rates of
inactivation and recovery from inactivation are not altered by
verapamil suggests that the crossover does not occur from
changes in the kinetics of channel inactivation
properties.33
The recovery of HERG current from verapamil block was
multiexponential, as shown in Figure 5. Because at
physiological pH verapamil (pKa 8.8)
exists in both neutral and charged forms, one possibility for this is
that the rapidly recovering component represents the unbinding
from the channel protein of the membrane-permeable neutral form of
verapamil, whereas the slowly recovering component
represents the charged moiety of verapamil trapped
in the channel pore. This is supported by the observation that recovery
of HERG current at –80 and –110 mV from block by the
membrane-impermeable N-methyl-verapamil analogue
is dominated by a slow time constant similar to that found with the
slow recovery process obtained with verapamil. The presence
of a rapid component of recovery at –50 mV with
N-methyl-verapamil is consistent with
rapid drug unbinding due to the increased probability of HERG channel
gating at this voltage.13 The voltage dependence of
the slow component of recovery can be explained by activation trapping
of the charged moiety of verapamil and of
N-methyl-verapamil in HERG channels after
channel closure, with hyperpolarization favoring
closure of the activation "gate." In the trapping hypothesis, the
activation gate must open before the charged drug can unbind, and less
negative recovery potentials increase the likelihood that the
activation gate will open transiently and release the charged drug. At
more negative voltages the probability of channel opening is reduced,
and the likelihood of drug remaining in the channel pore is increased.
Comparable findings have been shown with
tetraethylammonium and its
derivatives, which block K+
channels,34 35 and with analogues of lidocaine and
disopyramide, which block Na+
channels.22 36 37
Verapamil unblocking after repolarization permits HERG channels to become available for opening. This process would be expected to be faster at more physiological temperatures13 and may be an important pharmacological property. Most other drugs that block HERG channels, including class III antiarrhythmic agents, show minimal unbinding even with drug washout.25 38 39 Thus, the frequency-dependent effects of verapamil on HERG current result from use-dependent drug binding during cell depolarization as well as from time- and voltage-dependent drug unbinding with repolarization.
Intracellular Location of Phenylalkylamine Binding Domain
of HERG Channels
Several lines of evidence suggest that the verapamil
binding domain is located at a site accessible from the inside of the
cell. First, N-methyl-verapamil blocked HERG
current only when applied intracellularly. Second, varying
pHO (6.4 to 8.4), which changes the proportion of
drug in the membrane-permeable neutral form, altered block by
extracellularly applied verapamil. Third,
verapamil competes with the methanesulfonanilide
antiarrhythmic drug dofetilide for block of HERG channels. Dofetilide
is thought to bind to HERG channels near the internal mouth of the
channel pore. In addition, the Ser620Thr mutation, which is near the
internal mouth of the pore, decreased verapamil affinity
20-fold more than occurred with the Ser631Ala mutation, which is near
the external mouth of the pore.
Our experiments show that verapamil binds with little or no affinity to the closed state, but with high affinity to the channel during depolarization when the channel is either open or inactivated. The Ser620Thr and Ser631Ala mutations interfere with C-type inactivation. The finding of reduced block by verapamil in these mutants supports the concept that C-type inactivation plays an important role in high-affinity drug binding to HERG channels.24 25 26 Previous reports, however, have suggested that the extent of C-type inactivation may not parallel drug sensitivity. Wang et al26 reported that interfering with the development of C-type inactivation in HERG channels using high extracellular K+ did not have the same effect on drug affinity as did mutations that removed C-type inactivation. Ficker et al24 showed that mutation to a serine residue in bovine EAG channels in a position equivalent to HERG Ser620 enhanced dofetilide binding but did not produce inactivation. Our data with the Ser620Thr and Ser631Ala mutations show that the Ser620Thr mutation decreased verapamil affinity for HERG channels 20-fold more than occurred with the Ser631Ala mutation. One explanation for our results is that the decrease in high-affinity drug binding found with the mutated channels may be caused indirectly by interfering with the channel protein conformation associated with intact C-type inactivation, which may then alter drug binding affinity or restrict drug access to its binding domain. In addition, the mutation to the serine in position 620 may decrease further verapamil binding by altering a residue directly involved in its binding site similar to that proposed for dofetilide.24 An alternative explanation is that incomplete inactivation, which has been reported for the Ser631Ala mutation,40 41 might account at least partially for its increased verapamil sensitivity. Clearly, structural information about the HERG channel and drug binding to it would help to resolve these questions. Finally, it should be recognized that the mechanism of HERG channel block by verapamil may not be identical for both the charged and uncharged forms of the drug molecule.
Clinical Significance
Verapamil exerts effects on cardiac
arrhythmias that can be distinguished from those of other
calcium channel antagonist drugs. Some forms of
ventricular tachycardia are sensitive to
verapamil,6 7 it prolongs human atrial
refractoriness,9 and it may modify electrical remodeling
in atrial fibrillation or rapid pacing.8 9 Although the
antiarrhythmic efficacy of verapamil has usually been
ascribed solely to its suppression of L-type Ca2+
channels, our findings suggest that block of HERG channels also may
contribute to the cardiac effects of verapamil.
Suppression of IKr, or HERG channels, prolongs action potential duration and increases the refractory period and thereby may terminate reentrant circuits.14 This also can lead to the development of long-QT syndrome.14 Verapamil, which does not cause long-QT syndrome, may counteract the potential of HERG channel block–induced QT prolongation and early afterdepolarization generation through its block of L-type Ca2+ channels.42 Verapamil also unbinds from HERG channels at voltages near the resting potential, which may contribute to diminished HERG channel block. Finally, verapamil can cause bradycardia. Although this has been ascribed to block of inward Ca2+ current, suppression of IKr has also been reported to depress pacemaking.43
Conclusion
Verapamil potently blocks HERG
K+ channels expressed in HEK 293 cells. Diltiazem
weakly blocks HERG current, whereas nifedipine does not
exert an antagonist effect. Verapamil block was
use and frequency dependent. Verapamil appears to enter the
cell in its neutral form to bind to the channel protein at a site
accessible from the inside of the cell. Although intact C-type
inactivation seems important for highest-affinity verapamil
block, block still can occur in the absence of apparent C-type
inactivation, and the serine residue at position 620 may participate
directly in verapamil binding. These findings suggest that
verapamil shares HERG channel blocking properties with some
newer class III antiarrhythmic drugs, and these findings provide new
insights to explain some of the diverse effects exerted by
Ca2+ channel antagonist drugs on
cardiac arrhythmias.
|
Acknowledgments |
|---|
This work was supported, in part, by the Oscar Rennebohm Foundation and HL 56441. S.Z. is supported by a postdoctoral fellowship award from the American Heart Association, Wisconsin Affiliate. Z.Z. is a recipient of an American Heart Association Scientist Development Grant. We thank Drs Gail A. Robertson, Timothy J. Kamp, and Vicken R. Vorperian for helpful discussion.
Received January 15, 1999; accepted February 25, 1999.
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References |
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V. Rybalchenko, N. Prevarskaya, F. Van Coppenolle, G. Legrand, L. Lemonnier, X. Le Bourhis, and R. Skryma Verapamil Inhibits Proliferation of LNCaP Human Prostate Cancer Cells Influencing K+ Channel Gating Mol. Pharmacol., June 1, 2001; 59(6): 1376 - 1387. [Abstract] [Full Text]
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S. Zhang, S. Rajamani, Y. Chen, Q. Gong, Y. Rong, Z. Zhou, A. Ruoho, and C. T. January Cocaine Blocks HERG, but Not KvLQT1+minK, Potassium Channels Mol. Pharmacol., April 16, 2001; 59(5): 1069 - 1076. [Abstract] [Full Text]
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D. Thomas, G. Wendt-Nordahl, K. Röckl, E. Ficker, A. M. Brown, and J. Kiehn High-Affinity Blockade of Human Ether-A-Go-Go-Related Gene Human Cardiac Potassium Channels by the Novel Antiarrhythmic Drug BRL-32872 J. Pharmacol. Exp. Ther., April 12, 2001; 297(2): 753 - 761. [Abstract] [Full Text]
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 B.-R. Choi, T. Liu, and G. Salama The Distribution of Refractory Periods Influences the Dynamics of Ventricular Fibrillation Circ. Res., March 16, 2001; 88 (5): e49 - e58. [Abstract] [Full Text] [PDF]
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S. Fareh, A. Benardeau, and S. Nattel Differential efficacy of L- and T-type calcium channel blockers in preventing tachycardia-induced atrial remodeling in dogs Cardiovasc Res, March 1, 2001; 49(4): 762 - 770. [Abstract] [Full Text] [PDF]
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A. A. Grace and A.J. Camm Voltage-gated calcium-channels and antiarrhythmic drug action Cardiovasc Res, January 1, 2000; 45(1): 43 - 51. [Full Text] [PDF]
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D. M Roden and J. R Balser A plethora of mechanisms in the HERG-related long QT syndrome: Genetics meets electrophysiology Cardiovasc Res, November 1, 1999; 44(2): 242 - 246. [Full Text] [PDF]
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