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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
JPET 281:1247–1256, 1997
Vol. 281, No. 3
Printed in U.S.A.
Characterization of Nifedipine Block of the Human Heart
Delayed Rectifier, hKv1.51
XUE ZHANG, JAMES W. ANDERSON and DAVID FEDIDA
Department of Physiology, Botterell Hall, Queen’s University, Kingston, Ontario, Canada
Accepted for publication February 10, 1997
time constants (t2) for nifedipine block of hKv1.5 were concentration and voltage dependent. At 140 mV, t2 was 16.7 6 0.8
(10 mM), and 4.8 6 0.6 msec (50 mM), (n 5 4 – 8). Using a first
order kinetic analysis, apparent binding constants were 5.64 3
106 M21 s21 (k11, on-rate) and 37.5 s21 (k21, off-rate), with a
Kd of 6.65 mM, close to that obtained from the dose-response
curve. An increase in the off-rate (k21) could explain relief of
block .120 mV. The rank order of block under different patch
configurations was whole-cell ' outside-out . inside-out ..
cell-attached macropatches. Together, these suggested a
binding site for nifedipine at the extracellular pore of hKv1.5 or
at a hydrophobic channel domain within the lipid bilayer at a
site that is more accessible from the extracellular side.
Nifedipine is a Ca11 channel antagonist widely used for
the treatment of a variety of cardiovascular disorders. Normally, nifedipine blocks voltage-gated calcium channels with
a high affinity (Kd 5 310 nM in rabbit right atrium, Mecca
and Love, 1992; and 200 nM in myocardium, Charnet et al.,
1987). However, voltage-dependent K1 channels belong to
the same supergene family as Ca11 and Na1 channels (Catterall, 1988; Jan and Jan, 1989), with areas of homology in
the pore and around the carboxyl terminal of S6 in Ca11 and
K1 channels (Rampe et al., 1993; Nakayama et al., 1991), and
it is known that three types of Ca11 antagonists, verapamil,
nifedipine and diltiazem, all block cloned K1 channels
(Rampe et al., 1993; Grissmer et al., 1994). Verapamil and
nifedipine produced a marked block of the transient outward
current, Ito in rat ventricular myocytes (Jahnel et al., 1994).
Because Ito may contain multiple components of rapidly inactivating and slowly inactivating K1 channels, it is unclear
which specific Kv channel or channels may be blocked.
One component of Ito may be the rapidly activating delayed
rectifier K1 channel, hKv1.5 (Van Wagoner et al., 1996),
cloned from human heart (Tamkun et al., 1991; Fedida et al.,
1993), which is important in determining the duration of the
plateau phase of the cardiac action potential. Data exist
showing that hKv1.5 is blocked by all three types of Ca11
antagonists. Verapamil block of hKv1.5 was described in
detail (Rampe et al., 1993) and a mechanism of open channel
block from the inner pore was suggested. Diltiazem and
nifedipine (Grissmer et al., 1994) have also been shown to
block hKv1.5, but the detailed characteristics of nifedipine’s
effects on hKv1.5 have not been studied. Our study was
undertaken to examine the effects of nifedipine on hKv1.5
and to explore its mechanisms of action. Our data suggested
that nifedipine was an open channel blocker, which acted
predominantly at the external pore of hKv1.5 channels. The
effects of nifedipine were concentration and voltage dependent, but the block was somewhat relieved at more positive
potentials. This effect is in contrast to the block of hKv1.5 by
verapamil (Rampe et al., 1993), the inhibition of native K1
channels by nifedipine (Jacobs and DeCoursey, 1990) or
D600 and related phenylalkylamines (DeCoursey, 1995), and
the block of Ca11 channels by organic Ca11 channel antagonists (Sanguinetti and Kass, 1984; Hume, 1985; Uehara and
Hume, 1985), where block increased with potential.
Materials and Methods
Received for publication November 21, 1996.
1
This work was supported by grants from the Medical Research Foundation
of Canada, and the Heart and Stroke Foundation of Ontario, to D.F.
Cell culture. The methods used to establish stable HEK cell lines
expressing the hKv1.5 K1 channel and those used for electrophysi-
ABBREVIATIONS: W/C, whole cell recording; C/A, cell-attached recording; O/O, outside-out recording; I/O, inside-out recording; Q, gating
charge; HEK, human embryonic kidney; NMG, N-methyl-D-glucamine.
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ABSTRACT
Nifedipine antagonizes L-type Ca11 channels found throughout the cardiovascular system, but also blocks Kv channels,
which are members of the same supergene family. We have
examined nifedipine actions on the human heart K1 channel
(hKv1.5) expressed in human embryonic kidney cells. Peak and
steady-state currents on depolarization were reduced by nifedipine with Kd values of 18.6 6 2.7 and 6.3 6 0.5 mM respectively at 140 mV, and with Hill coefficients of 0.75 6 0.04 and
0.93 6 0.03. Block increased rapidly between -10 mV and 110
mV, coincident with channel opening and suggested an open
channel block mechanism, which was confirmed by tail current
crossover on repolarization (unblock on channel closing). At
more positive potentials than 120 mV, block was relieved. The
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Zhang et al.
hKv1.5 channels in HEK cells, there was no need for signal averaging, and single gating current transients could be observed. The
average cell capacitance was quite small, and the absence of ionic
current at negative membrane potentials allowed faithful leak subtraction of data.
Data analysis. The concentration-response curve (figs. 2C and
7B) for changes in peak and steady-state current produced by nifedipine were computer-fitted to the Hill equation:
f 5 1/@1 1 ~Kd/[D]n)]
(1)
where f is the fractional current block (f 5 1-Idrug/Icontrol) at drug
concentration [D]; Kd is the concentration producing half-maximal
inhibition and n is the Hill coefficient. The rapid component of
inactivation induced by nifedipine was much faster than that observed in the absence of drug. Therefore, we used this drug induced
time-constant (t2) as an approximation of the drug channel interaction kinetics, as described previously (Snyders et al., 1992; Slawsky
and Castle, 1994), according to the equations:
1/t2 5 k11[D] 1 k21
(2a)
Kd 5 k21/k11
(2b)
and
in which t2 is the current decay time constant caused by the drug; [D]
is the concentration of drug; k11 and k21 are the apparent rate
constants of binding and unbinding for the drug, respectively.
The voltage dependence of block for the uncharged drug was
determined as follows: leak-corrected current in the presence of drug
was normalized to matching control at each voltage above -20 mV.
Using data points in the range of full channel opening (.120 mV),
the voltage dependence of block was fitted to a linear equation, and
the slope of block determined. Alternatively, in “Discussion,” we have
calculated the fractional block (f 5 1-Inif/Icontrol) at each potential and
fitted data to the Woodhull equation:
f 5 [D]/([D] 1 K*d z e2dzFE/RT)
(3)
where F, R, z and T have their usual meanings, d represents the
fractional electrical distance, i.e., the fraction of the transmembrane
electrical field sensed by a single charge at the receptor site. K*d
represents the binding affinity at the reference voltage (0 mV).
Experimental values are given as means 6 S.E.. Analysis of variance was used to compare the effects of nifedipine on hKv1.5 currents under different macropatch configurations; Paired t test was
used to compare the amplitudes of off-gating charge in the presence
of nifedipine with control. A value of P , .05 was considered statistically significant.
Results
Concentration-dependent and reversible block. The
data in Figure 1 show the effects of nifedipine on hKv1.5
currents expressed in HEK cells under W/C recording conditions. The cell was held at -80 mV, and membrane currents
were elicited before exposure to nifedipine, during exposure
to 10 mM and 50 mM nifedipine and after wash out. These are
currents in response to a series of step depolarizing pulses
from -30 mV to 140 mV. Extracellular application of 10 mM
and 50 mM nifedipine resulted in a reduction of both peak
and steady-state hKv1.5 currents with a marked increase in
the rate of outward current relaxation in the presence of
nifedipine (Fig. 1B, 1C). This meant that steady-state currents recorded at the end of 250 ms pulses, were much more
reduced by nifedipine than the peak currents, in a concentration-dependent manner. After 5 min wash out to control
bath solution, the effect of nifedipine was largely reversed.
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ological measurement of hKv1.5 currents have been described in
detail previously (Fedida et al., 1993). Alternatively, hKv1.5 was
transiently transfected into HEK cells using the mammalian expression vector pRc/CMV. Cells expressing hKv1.5 were detected by
cotransfecting cells with the vector pHook-1 (Invitrogen, San Diego,
CA). This plasmid encoded the production of an antibody to the
hapten phOX, which when expressed is displayed on the cell surface.
Transfected cells were maintained in modified Eagle’s medium at
37°C in an air/5% CO2 incubator in 25-mm Petri dishes plated on
glass coverslips until use. One hour before experiments, cells were
treated with beads coated with phOX. After 5 min, excess beads were
washed off with cell culture medium and cells which had beads stuck
to them were used for electrophysiological tests. The efficiency of
dual transfection was observed to be better than 80%, so the beads
provided a good means of identifying those cells that expressed
hKv1.5. No difference was observed from data obtained using stable
cell lines or transient expression of hKv1.5, so all results have been
included in the analysis.
Solutions. For W/C and O/O macropatches, the control pipette
filling solution contained (in mM): KCl, 130; EGTA, 5; MgCl2, 1;
HEPES, 10; Na2ATP, 4; GTP, 0.1; and was adjusted to pH 7.2 with
KOH. The control bath solution contained (in mM): NaCl, 135; KCl,
5; sodium acetate, 2.8; MgCl2, 1; HEPES, 10; CaCl2, 1; and was
adjusted to pH 7.4 with NaOH. For C/A and I/O macropatches, the
pipette filling solution was the extracellular control solution used in
W/C recording, and the bath solution was a 135 mM K1 solution
designed to zero the membrane potential (in C/A recording). It contained (in mM): KCl, 135; HEPES, 10; MgCl2, 1; dextrose, 10; and
was adjusted to pH 7.4 with KOH. For gating current experiments,
cells were superfused with a solution containing (in mM): NMG, 140;
HEPES, 10; CaCl2, 1; MgCl2, 1; dextrose, 10; pH 7.4 with HCl. The
pipette solution contained (in mM): NMG, 140; HEPES, 10; MgCl2, 1;
EGTA, 10; pH 7.2 using HCl. Nifedipine was dissolved in alcohol at
a stock concentration of 1, 10 or 100 mM, and was protected from the
light during all experiments. After control data were collected, the
bathing solution was changed to include nifedipine. Nifedipine effects were very rapid on cells, apparently reaching a steady-state
within three pulses (30 sec). Steady-state measurements made in the
presence of nifedipine were obtained after at least 3 min exposure to
the drug. All chemicals were from Sigma Chemical Co. (St. Louis,
MO).
Electrophysiological procedures. Coverslips containing cells
were removed from the incubator before experiments and placed in a
superfusion chamber (volume 250 ml) containing the control bath
solution at 22 to 23°C. W/C, C/A, O/O and I/O macropatch recordings
were made via the variations of the patch-clamp technique (Hamill et
al., 1981), using an Axopatch 200A amplifier (Axon Instruments,
Foster City, CA). Patch electrodes were pulled from thin-walled
borosilicate glass (World Precision Instruments, Sarasota, FL) on a
horizontal micropipette puller, fire-polished, and filled with appropriate solutions. Electrodes had resistances of 1.5 to 3.0 mV when
filled with control filling solution. Analog capacity compensation and
75 to 85% series resistance compensation were used in all W/C
measurements. In some experiments, leak subtraction was applied
to data. Membrane potentials have been corrected, where appropriate, for junctional potentials that arose between the pipette and bath
solution. Data were filtered at 5 to 10 kHz before digitization and
stored on a microcomputer for later analysis using the pClamp6
software (Axon Instruments). In experiments where gating currents
were recorded, the sample rate was 330 kHz and currents were
usually leak-subtracted using a P/6 protocol similar to that described
previously (Stühmer et al., 1991; McCormack et al., 1994; Bouchard
and Fedida, 1995). Currents were low-pass filtered at 10 to 50 kHz
during data collection or later at 10 kHz for data presentation.
Pipettes were routinely sylgarded and fire polished to reduce electrode capacitance and improve seal resistance. The system that we
used for expression of hKv1.5 conferred certain advantages for the
measurement of gating current. Due to the high level of expression of
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1997
Figure 1D showed that the current was restored to 85% of
control current level. Current-voltage (I-V) relationships for
the peak and steady-state outward current in the absence,
presence of 10 and 50 mM and washout of nifedipine are
shown in Figure 1E and F. Here it can be seen that both peak
and steady-state current were reduced in a concentrationdependent manner, but that the reduction of steady-state
current was much more than that of peak current. Native
HEK cells also possess a small outwardly rectifying K1 current. The mean amplitude of this current was 204 6 19.7 pA
at 140 mV (n 5 10), which is less than 2% of the total current
in transfected cells on depolarization (compare control
hKv1.5 current amplitudes in figs. 1E and 2). Nifedipine also
reduced this current, with 50% block at .20 mM (n 5 3).
However, due to the small current size, we have not considered contamination by this endogenous current to be significant.
The effects of nifedipine on hKv1.5 over a wide range of
concentrations from 0.2 to 200 mM, further confirmed that
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the block was concentration-dependent (fig. 2). Low concentrations of nifedipine (between 0.2 and 10 mM) are shown in
figure 2A. The threshold for nifedipine action on hKv1.5 was
around 100 to 200 nM, which was somewhat lower than
expected for dihydropyridine block of hKv1.5 (cf., Grissmer et
al., 1994). At low concentrations, up to '1 mM, there was
little effect on peak outward current, but nifedipine induced
a slow decay of current that reached a steady-state at the end
of 1 sec depolarizing voltage clamp pulses. At higher concentrations than 1 mM, a reduction of peak current was observed
and a more rapid decay of current to the steady-state. At high
concentrations of nifedipine a marked reduction of peak current was observed (fig. 2B) with rapid current decay to the
steady state. The concentration response curve for block of
peak and steady-state outward current by nifedipine at a test
potential of 140 mV is shown in figure 2C. As expected from
data in figures 1 and 2, steady-state currents (E) showed a
greater level of block at any particular nifedipine concentration than peak currents (F). The solid lines were fit to the
data using the Hill equation (see “Materials and Methods,”
equation 1). The resultant Kd values for the peak and steady
state hKv1.5 current block by nifedipine were 18.6 6 2.7 and
6.3 6 0.5 mM, the Hill coefficients were 0.75 6 0.04 and
0.93 6 0.03, respectively.
Voltage-dependent block. To examine the voltage-dependence of block, the relative steady-state current Inif/Ictl at
the end of 400 msec voltage clamp pulses was plotted as a
function of potential. The data in figure 3 show normalized
hKv1.5 current-voltage(I-V) relationships for different concentrations of nifedipine. The dotted line is the normal activation curve of hKv1.5. This was obtained from the deactivating tail current amplitude at -20 mV after 15 msec
depolarizing steps to potentials between -80 to 1100 mV
from a holding potential of -100 mV. In the presence of 5, 10,
20 and 50 mM nifedipine, block increased rapidly between -10
and 110 mV, coinciding with the voltage range of channel
opening. These data suggest that nifedipine binds primarily
to the open state of hKv1.5 channels. Over the voltage range
between 120 and 190 mV, almost all channels are open, but
block was slightly relieved with depolarizing test potentials
and showed a shallow voltage dependence. At different concentrations of nifedipine, block at 190 mV compared with
120 mV was reduced from 0.50 6 0.01 to 0.40 6 0.03 (5 mM),
from 0.57 6 0.04 to 0.49 6 0.06 (10 mM), from 0.70 6 0.05 to
0.60 6 0.05 (20 mM), and from 0.89 6 0.02 to 0.84 6 0.02 (50
mM); (n 5 4–6). Over the potential range where channels
were fully open, the relationship for block by nifedipine at
depolarizing voltages was well fitted to a linear equation. The
slopes of fit were 1.53 3 1023, 1.16 3 1023, 1.38 3 1023 and
7.35 3 1024 mV21 with 5, 10, 20 and 50 mM nifedipine,
respectively.
Nifedipine has a low pKa # 1.0 (Uehara and Hume, 1985),
so almost all of the drug is uncharged in the physiological pH
range around 7.4. In this situation a voltage-dependence to
the drug action (once all channels are open) was not expected.
The voltage dependence was not consistent with a positively
charged drug acting at the intracellular mouth of the channel. One would expect increased block with potential as has
been shown for the action of quinidine (Snyders et al., 1992;
Fedida, 1997), quinine or tetrapentylammonium ions (Snyders and Yeola, 1995) on hKv1.5. However, if drug binding
was coupled in some way to a charged process, like K1 flux,
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Fig. 1. Nifedipine block of hKv1.5 current. A to D, Currents were
elicited by 250-msec depolarizing pulses from the holding potential of
-80 mV to voltages between -30 mV and 140 mV in increments of 110
mV. W/C currents were recorded in the absence of nifedipine (A) and
the presence of 10 mM (B), 50 mM (C) and after 5 min wash out (D)
nifedipine, respectively. E and F, Current-voltage (I-V) relationships for
peak and steady-state outward current in the absence (F) and presence of 10 (É), 50 (ç)mM and wash out (M) of nifedipine are shown in E
and F, respectively. The dotted line marks the zero current level. Data
were from the same cell as panels A to D. Note that after exposure
to high concentrations of nifedipine, wash out was often incomplete
as in D.
Nifedipine Block of hKv1.5
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Zhang et al.
Vol. 281
and if the drug had an extracellular site of action, the results
would be consistent with a relief of block with intracellular
depolarization. This point is developed further in “Discussion.”
Effects of nifedipine on current decay of hKv1.5. In
the absence of nifedipine, current decayed slowly (fig. 2, A
and B) and during the relatively short voltage pulses used
here, was fitted to a single exponential function with a decay
time constant of 230 6 8 msec (n 5 14) at 140 mV. After
addition of nifedipine, the rate of current decay increased in
a concentration-dependent manner (fig. 2, A and B) and could
be well fitted with a double exponential function. The
nifedipine-induced fast time constant, t2, was used as an
index of the rate of block of hKv1.5. Figure 4A shows the
effects of different concentrations of nifedipine and different
voltages on the mean t2 values. At each voltage, t2 decreased
in a concentration-dependent manner, which indicated a
more rapid current decay. At each concentration of nifedipine, t2 became faster at voltages between 0 and 130 mV,
then t2 showed a shallow decrease at more positive potentials.
The rapid component of inactivation induced by nifedipine
(t2, fig. 4A, 5–50 mM) was much faster (approximately 10-fold
or more) than the slow component observed in the absence of
drug. Therefore, t2 was used as an approximation of the time
course of drug-channel interaction, as described previously
(Snyders et al., 1992). Figure 4B shows the plot of 1/t2 vs. the
concentration of nifedipine at a test potential of 140 mV.
From equation 2a (see “Materials and Methods”), the best
least squares fit to the data resulted in an apparent associ-
Fig. 3. Voltage-dependence of current block by nifedipine. The dotted
line represents the activation curve of control hKv1.5. This was obtained from deactivating tail current amplitude at -20 mV after 15-msec
depolarizing steps to potentials between -80 to 1100 mV, from a
holding potential of -100 mV. The symbols denote ratios of steady-state
current in nifedipine to the control current value at each potential. Open
symbols show the data between -10 and 110 mV. Filled symbols show
the data at voltages positive to 120 mV. The solid lines were the best
fit to a linear function in the form of y 5 ax 1 b. With 5, 10, 20 and 50
mM nifedipine, the slopes from this linear fit were 1.53 3 1023, 1.16 3
1023, 1.38 3 1023 and 7.35 3 1024 mV21. Data were mean 6 S.E. from
4 to 10 experiments.
ation rate constant, k11 of 5.64 3 106 M21 sec21 and an
apparent dissociation rate constant, k21 of 37.5 sec21. The
resultant Kd value from equation 2b (see “Materials and
Methods”) was 6.65 mM, which is consistent with the Kd
value of 6.3 mM from the dose-response curve (fig. 2B).
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Fig. 2. Concentration-dependent block of hKv1.5 by nifedipine. A and B, W/C current recording during voltage
pulses to 140 mV from the holding potential of -80 mV.
Currents were recorded in the steady-state at a large
range of nifedipine concentrations. Current traces in A
and B were from two different cells. The cell in A was
exposed to low concentrations of nifedipine from 0.2 to 10
mM and the cell in B was exposed to high concentrations
of nifedipine from 20 to 200 mM. C: Dose-response curve
for nifedipine block of hKv1.5. Peak and steady-state
reduction of current (relative to control) at a test potential
of 140 mV was plotted against the concentration of nifedipine. Data were averaged from 4 to 10 experiments.
Solid lines were fit to the data using a Hill equation (see
“Materials and Methods,” equation 1). For the reduction of
peak current, the Kd value was 18.6 6 2.7 mM, Hill coefficient was 0.75 6 0.04; for the reduction of steady-state
current, the Kd value was 6.3 6 0.5 mM, and the Hill
coefficient was 0.93 6 0.03.
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Nifedipine Block of hKv1.5
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Fig. 4. Time constants of hKv1.5 current decay in the presence of
nifedipine. A, Voltage-dependence of hKv1.5 current decay in different
concentrations of nifedipine. In the presence of nifedipine, t2 was the
fast component obtained from biexponential fits to the falling phase of
the currents obtained during 250-msec voltage steps to between 0 and
190 mV from the holding potential -80 mV. Data were mean 6 S.E.
from 4 to 10 experiments. B, Kinetics of nifedipine block of hKv1.5. The
reciprocal of the nifedipine-induced fast time constant (1/t2) at 140 mV
has been plotted against the nifedipine concentration. The best fit to
the data (solid line) using the equation 1/t25k11. [D] 1 k21 resulted in
an apparent association rate constant (k11) of 5.64 3 106 M21 sec21
and an apparent dissociation rate constant (k21) of 37.5 sec21. The Kd
value (k21/k11) was 6.65 mM. Error bars, S.E. from four to six experiments.
Open or closed channel block? Two methods are often
used to decide whether drugs predominantly interact with
open or closed channels. For open channel blockers that have
a slow block rate and that dissociate from closed channels,
currents peak in the presence of drug at a constant level,
before a rapid decay occurs with drug-channel interaction.
Such an effect is seen when K1 channels are exposed to
4-aminopyridine for the first time (Choquet and Korn, 1992;
Bouchard and Fedida, 1995). As a result, an acceleration of
Fig. 5. Concentration-dependent rate of onset of hKv1.5 block by
nifedipine. Original currents were obtained from a holding potential of
-80 mV during 200-msec pulses to a test potential of 140 mV. Nifedipine sensitive current was calculated from the subtraction of steadystate currents in control and the presence of nifedipine, divided by the
control current level (Ictl-Inif)/Ictl. These have been plotted against the
time from the start of the voltage clamp pulse. The times of onset of
block were 3.0, 2.4, 1.3, 1.1, 0.75 and 0.35 msec with 5, 10, 20, 50, 100
and 200 mM nifedipine. The solid lines were the best fits to a biexponential equation of the form [y 5 A1*exp(-t/t1) 1 A2*exp(-t/t2) 1 C]. Data
shown were from two different cells.
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current inactivation is generally inferred to mean open-channel binding (Slawsky and Castle, 1994). However, if the peak
current is reduced due to rapid drug-channel interaction, as
for tetrapentylammonium block of hKv1.5 (Snyders and
Yeola, 1995) or in our experiments with nifedipine, it can be
more difficult to exclude a resting channel block. One method
to analyze the onset of block is to fit the fractional block of
current back to the start of the depolarizing pulse (Slawsky
and Castle, 1994). If the fit intersects zero block after the
start of the pulse, a purely open channel block mechanism is
favored.
The time course for the development of inhibition by nifedipine is shown in figure 5. The drug-sensitive current expressed as a proportion of the outward current observed in
the absence of the drug ((Ictl-Inif)/Ictl) has been plotted as a
function of time after the start of depolarization (at t 5 0
msec). Inhibition increased in an exponential manner with
time and it can be seen that both the maximum inhibition
and the rate of development of this inhibition were concentration dependent. The voltage pulse was applied at 0 msec
and the exponential onset of current block always occurred
after this time. The mean times of onset of current block after
application of the depolarizing clamp pulse were 3.33 6 0.18,
2.80 6 0.14, 1.56 6 0.19, 1.09 6 0.25, 0.65 6 0.35 and 0.42 6
0.30 msec with 5, 10, 20, 50, 100 and 200 mM nifedipine (n 5
3–5). At a concentration of 200 mM nifedipine, inhibition was
apparent almost immediately on depolarization, as indicated
from the time of intersection of the fit line with the abscissa.
Clearly, block occurred with time constants not dissimilar
from the time constants of current activation at the higher
concentrations. These data support the idea that nifedipine
binds to open channels, but it is difficult at high concentrations to exclude nifedipine binding to steps in the activation
pathway during the early phase of channel opening.
This open channel block was further confirmed by crossover of tail currents in the absence and presence of nifedipine. Short, 100-msec depolarizing pulses were given to fully
open hKv1.5 channels and cells were then repolarized to a
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Zhang et al.
Vol. 281
potentials of -50 mV to measure outward tail currents from
deactivating channels (fig. 6A, inset). A typical example of
currents is illustrated in figure 6A. In the presence of increasing concentrations of nifedipine, peak currents were
reduced and current decay during voltage clamp pulses was
accelerated. The tail currents from figure 6A are enlarged in
figure 6B and C. In the presence of nifedipine, the initial tail
current amplitude was less (slowly decaying tracings) compared with control, and there was an obvious rising phase
that can be seen at the beginning of the tail current in the
presence of 10 and 5 mM nifedipine, but not obvious with 2
mM nifedipine and absent in control (fig. 6C, lower panel).
Then tail currents crossed over each other (fig. 6C, upper
panel). Results of this nature suggest that nifedipine dissociates from deactivating channels rather slowly compared
with control channel deactivation, and that nifedipine must
dissociate from its binding site before channels can close. All
these properties were consistent with an open channel action
of nifedipine.
Site of action. Nifedipine is a 1,4-dihydropyridine with a
pKa # 1.0 (Uehara and Hume, 1985). Thus, at a physiological
pH of 7.4, almost all of the nifedipine will be in its neutral
form. As an uncharged moiety it should rapidly cross biological membranes, and thus it has a possible site of action at
the outer or inner mouth of open hKv1.5 channels. It has
been reported that nifedipine can block Ca11 channels from
the extracellular side or penetrate the membrane to approach its binding site in the hydrophobic domain near to the
extracellular side. To attempt to determine on which side of
the membrane that hKv1.5 channels could be blocked by
nifedipine, the efficacy of block under different macropatch
recording conditions was compared. The data in figure 7A
illustrate the effects of 10 and 100 mM nifedipine on the
current recorded from O/O and C/A macropatches. In both
cases the currents were recorded in the steady state in nifedipine, after at least 3 min exposure. In the presence of
nifedipine, at similar concentrations, current was much reduced in the O/O patch mode of recording compared with the
C/A patch. Summary steady-state data from two to eight cells
in figure 7B shows the effect of different concentrations of
nifedipine on hKv1.5 in C/A, I/O, O/O macropatches and
during W/C recording. Data points were obtained by comparing the steady-state current in control and different concentrations of nifedipine under the different recording conditions. Compared with O/O macropatches and W/C recording,
the current in I/O and C/A macropatches was relatively less
sensitive to nifedipine. At concentrations of 1 to 5 mM nifedipine, very little block was observed in C/A macropatches,
whereas at 5 mM nifedipine, current was '50% blocked in
W/C and O/O macropatch recordings. The rank order of block
of hKv1.5 by nifedipine was then, whole-cell ' outside-out .
inside-out .. cell-attached macropatches. Analysis of variance of the results with 10, 20, 50 and 100 mM nifedipine
showed that the difference of relative current between C/A or
I/O macropatches and O/O macropatches or W/C recordings
was statistically significant (P , .05), the difference between
O/O macropatches and W/C recording was not statistically
significant (P . .05).
Our rationale for determining the likely site of action of
nifedipine is based on our assumption that variations in the
efficacy of block by nifedipine are closely related to the local
concentrations of nifedipine under different recording condi-
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Fig. 6. Deactivation tail current crossover caused by nifedipine. A, Under W/C recording conditions, cell was
held at -80 mV and stepped to 140 mV for 100 msec,
then stepped back to -50 mV as shown by the protocol
inset in A. Tail currents were recorded during the last
voltage step to -50 mV. Superimposed currents are
shown in control and the presence of 2, 5 and 10 mM
nifedipine. B, Deactivation tail currents from panel A (dotted box) were enlarged to illustrate cross-over of control
and in the presence of 2, 5 and 10 mM nifedipine. C, Tail
currents in B were enlarged again to show crossover
clearly (upper panel, in the presence of 5 mM nifedipine)
and the rising phase at the beginning of tail currents
(lower panel) in the presence of 2, 5 and 10 mM nifedipine,
with the control tracing for comparison.
1997
tions. If the binding site of nifedipine is at or near to the
extracellular side, during W/C recording when nifedipine was
superfused into the bath, it can bind to its receptor directly
and efficiently. For the C/A macropatches, when nifedipine is
added to the bath, it has to enter the cell, cross the patch and
block from the pipette side. As the pipette filling solution
contains no nifedipine, it provides a large volume into which
nifedipine at the extracellular face of the patch would be
rapidly diluted away by the pipette constituents. This is
likely to reduce the effective local concentration of nifedipine
at the extracellular side of the patch in the C/A configuration
and thus decrease the efficacy of nifedipine block of hKv1.5.
A similar explanation can be applied to the results from I/O
1253
(decreased efficacy of block) and O/O (high efficacy of block)
macropatch configurations. In confirmation of these results
nifedipine added to the pipette filling solution had little effect
on whole cell currents.
An important characteristic of open channel blocking
drugs that act on voltage-gated Kv channels at the internal
face of the membrane is that they usually cause channel
gating charge immobilization. This has been shown to be true
for tetraethylammonium ions (Stühmer et al., 1991; Bezanilla et al., 1991), for 4-aminopyridine (Bouchard and Fedida,
1995; McCormack et al., 1994) and quinidine (Fedida, 1997).
It seems that the charged drug binding to sites at the inner
mouth of the pore somehow prevents some outward movement or rapid return of the voltage sensor on repolarization.
This leads to the reduction or slowed movements of different
components of gating charge. This may be because of their
charge that directly influences surface charge at the inner
mouth of the pore. Alternatively, the channels may be unable
to return to their resting conformation until the drugs dissociate, due to steric effects of the drugs hindering channel
closure. This latter mechanism of immobilization does not
depend on charged forms of the drugs. We have postulated
earlier that nifedipine exerts its action towards the extracellular surface, and thus we do not expect large effects on
hKv1.5 gating currents. The effect of different concentrations
of nifedipine on typical hKv1.5 gating currents is illustrated
in figure 8. Ionic currents were eliminated by substitution of
all permeant monovalent ions in the pipette and extracellular solutions with NMG (see “Materials and Methods”). In
transfected HEK cells, data corrected for leak revealed rapid
transient currents on depolarization and repolarization (fig.
8, A and C). The holding potential was -100 mV. The gating
currents on depolarization (on-gating current) reached their
peak between 2.2 and 0.5 msec, dependent on the pulse
potential. Note that this time is theoretically well within the
time resolution of the recording system that is limited by the
cell size and access characteristics. As noted previously, peak
gating current increased with depolarization, although total
charge moved (Qon) eventually saturated (Bezanilla et al.,
1991; McCormack et al., 1994; Bouchard and Fedida, 1995)
(fig. 8, E and F). The more rapid decay of on-gating current
underlies the increasingly rapid activation of ionic current
with larger depolarizations. The peak gating current on repolarization (off-gating current) was reduced and the decay of
off-gating current slowed for larger depolarizations, and this
also is known to occur in Shaker K1 channels (Stefani et al.,
1994). Upon repolarization, gating charge (Qoff) returns, and
although slower for large positive depolarizations, it was well
conserved, with the ratio Qoff/Qon usually close to 1.0. In the
presence of nifedipine (fig. 8, B and D), on and off-gating
currents were very similar to those seen in control at concentrations of nifedipine from 5 to 50 mM. However, mean data
(fig. 8, E and F) revealed a small reduction in off-gating
charge after depolarizations to more positive potentials than
0 mV at both 5 and 50 mM nifedipine. The reductions were
statistically significant at potentials positive to 0 mV (paired
t test, P , .05). This arises from an increased immobilization
of off-gating current at potentials at which channels are open
and suggests that nifedipine block of open hKv1.5 channels is
associated with some inhibition of conformational changes
that accompany channel closing. However, these changes are
very small compared with the actions of drugs at the intra-
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
Fig. 7. Comparison of nifedipine-induced block of hKv1.5 in different
recording configurations. A, For O/O macropatch (left panel), current
traces were elicited from the holding potential of -80 mV during a
400-msec pulse to the test potential of 140 mV. For the C/A macropatch (right panel), traces were obtained from the holding potential of
180 mV to a pipette test potential of -40 mV in a 200-msec pulse.
Traces shown were from two different cells in which currents were
recorded in the absence and presence of 10 and 100 mM nifedipine.
Note that nifedipine actions on hKv1.5 were stable after only two or
three pulses after nifedipine was washed into the bath. However, all
steady-state data shown here, and in B were obtained after more than
3 min exposure to nifedipine. B, The relative steady-state currents
(Inif/Ictl) in the presence of different concentrations of nifedipine have
been obtained from four modes of patch clamp recording. These were
C/A (F), I/O (E) and O/O (ç) macropatches, and W/C (É) recording.
Current were obtained during the same voltage protocol as illustrated in
A. Solid lines were the best fits to the data using Hill equation (see
“Materials and Methods,” equation 1). For the data from C/A, I/O, O/O
macropatches and W/C recording, the resultant Kd were 47.3 6 10.1,
7.5 6 1.5, 6.9 6 1.0, 6.7 6 0.6 mM and Hill coefficients were 0.88 6
0.05, 0.70 6 0.06, 0.90 6 0.06, 0.95 6 0.04, respectively. Data were
means 6 S.E. from three to seven experiments. The asterisk represents
a statistically significant (P , .05) difference between the C/A or I/O
macropatch data and O/O macropatch or W/C data.
Nifedipine Block of hKv1.5
1254
Zhang et al.
Vol. 281
cellular mouth of K1 channels, and seem unlikely to be
related to the ionic current block by nifedipine which has a Kd
of 6.3 mM at 140 mV (fig. 2C).
Discussion
We have investigated the mechanism of action of nifedipine on the hKv1.5 channel expressed in a human cell system. Nifedipine is a tissue-specific Ca11 channel antagonist
that has a major role in the vascular bed where it is an
effective vasodilator. In our study nifedipine also produced a
strong block of a human heart Kv channel.
Nifedipine blocks hKv1.5 with a low Kd. Nifedipine is
a widely used Ca11 antagonist and has been shown to block
native Ca11 channels with Kd in the range of 200 to 310 nM
(Charnet et al., 1987; Mecca and Love, 1992). Cloned Ca11
channels are also blocked by dihydropyridines and phenylalkylamines with Kds in the 100s of nM to low mM range
(Schuster et al., 1996). Nifedipine blockade of native cardiac
potassium channels has also been reported. Transient outward potassium currents It in rabbit atrium (Gotoh et al.,
1991) and Ito in rat ventricular myocytes (Jahnel et al., 1994)
were largely blocked by 30 mM nifedipine and the blockade
was concentration-dependent. Besides the block of potassium
channels in intact myocytes, nifedipine also caused the block
of cloned potassium channel hKv1.5 with a Kd of 81 mM
(Grissmer et al., 1994). Our data in figures 1 and 2 demonstrated that nifedipine accelerated the time course of decay of
hKv1.5 currents. Currents reached a peak that was less than
corresponding control currents at concentrations greater
than the Kd. Subsequently nifedipine caused a rapid current
decay that was concentration dependent. Such effects of nifedipine allowed us to calculate dose-response curves for both
the peak and steady-state outward currents (fig. 2). Fits of
the Hill equation to these data gave Kd values of 18.6 6 2.7
and 6.3 6 0.5 mM for peak and steady-state currents, respectively, with Hill coefficients of 0.75 6 0.04 and 0.93 6 0.03.
The peak current measurement represented the partial block
of current by nifedipine, and the interaction between rates of
channel opening and the onset of block at different pulse
potentials and nifedipine concentrations (see below). For this
reason, the Kd value (18.6 mM) was expected to be lower than
for steady-state block of open channels (6.3 mM). The nonsteady-state value for the Hill coefficient that was measured
(0.75) from the peak current dose-response cannot then be
used to indicate cooperativity. The measurement of steadystate current block reflected the equilibrated interaction of
hKv1.5 channels with nifedipine at different concentrations
and potentials and was of more interest in the present study.
The Hill coefficient close to 1.0 for steady-state current block
suggests that binding of one nifedipine molecule per channel
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Fig. 8. Effect of nifedipine on gating currents of
hKv1.5 channel. A and B, Data in panel A and B are
the gating currents of hKv1.5 channel in the absence
(A) and presence (B) of 5 mM nifedipine. Cell was
depolarized from -100 to 170 mV for 12 msec from a
holding potential of -100 mV to initiate the on-gating
current, then repolarized to -100 mV to record offgating current. Traces shown are from -70 to 130 mV
in increments of 120 mV. C and D, Data from a
different cell in the absence (C) and presence (D) of
50 mM nifedipine. Traces were elicited using the same
voltage protocol as in panels A and B. E and F,
Integrated on- (E) and off- (É) gating charge movement in the presence of 5 mM (E) and 50 mM (F)
nifedipine expressed relative to the maximum on- (F)
and off- (ç) gating charge movement in controls. Data
were means 6 S.E. from four (F) or six (E) experiments. The asterisk represents a statistically significant (P , .05) difference between off-gating
charge movement in control and in the presence of
nifedipine.
1997
1255
the dose response curve in figure 2C. It should be noted that
t2 represents the transition from the open to block state and
does not hold at small depolarizations (,0 mV), where activation is much slower, or at high drug concentrations, in
which case the time constant of block may be similar to that
of activation. In these cases the binding and unbinding rates
of nifedipine cannot be extracted from a simple model such as
that given above.
Voltage-dependence of block. In some K1 channels
voltage-dependence of nifedipine block has been described. In
rat alveolar-epithelial cells an inactivating delayed rectifier
K1 channel demonstrated a voltage-dependent tblock (Jacobs
and DeCoursey, 1990). In frog atrial myocytes, block of Ik by
nisoldipine, a nifedipine analogue was voltage dependent
(Hume, 1985). Our data in figure 3 showed that current was
blocked quickly by nifedipine in the voltage range of channel
opening. After channels were fully open, block was slightly
relieved at more positive voltages. This relief of block was
independent of the concentration of nifedipine between 5 and
50 mM (fig. 3), and was well fitted to a linear function (fig. 3).
The slopes of the fits were approximately similar, between
0.75 3 1023 and 1.5 3 1023 mV21, and these quantify the
increase in normalized current with depolarizations in the
presence of the drug. At the same time, data in figure 4A
indicate that once channels were fully open (.130 mV),
there was a shallow decrease in the time constants of block
with potential. From the above and a consideration of equation 2a and 2b, these data indicate that the voltage-dependence to the block must be given by an increase in the off-rate
(k21) rather than a decrease in the on-rate (k11) at more
positive potentials. There are a number of possible explanations for this. First, although nifedipine is uncharged, its
binding site may be coupled to some voltage-dependent process, such as conformational changes in the pore with potential or movement of the voltage sensor (Jacobs and DeCoursey, 1990), which can sense the transmembrane voltage
change and confer voltage-dependence to block by an uncharged drug. It has been proposed by De Coursey that
neutral phenylalkylamine drugs may have rapid access to
their receptors, where block is then stabilized by protonation
of the drugs (DeCoursey, 1995). Although nifedipine has a
much lower pKa, such a mechanism could explain the voltage-dependence observed in the present experiments.
Nifedipine site of action. An alternative explanation for
the voltage-dependence of block could be that nifedipine
blocks open K1 channels from an external site. It is possible
that at more positive voltages, potassium permeation increases and hinders in some manner the binding of nifedipine
to its site with a resultant relief of block. Analogous with this,
it is known that K1 occupancy of sites in the external mouth
of the K1 channel pore affects the rate at which charged
blockers (ions) can interact with the channel (Baukrowitz
and Yellen, 1996). We have established that nifedipine induced open-channel block of hKv1.5 and two additional lines
of evidence suggest that the site is in the external mouth of
the pore. The rank order of block described in figure 7 was
W/C ' O/O . I/O .. C/A macropatch. This suggested a
preferential nifedipine block of hKv1.5 channels from the
extracellular side or at a hydrophobic domain accessible from
the extracellular surface. A similar conclusion has been
drawn for native K1 channels (Jacobs and DeCoursey, 1990;
although see DeCoursey, 1995) and for the dihydropyridine
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is sufficient to block the hKv1.5 channel. Although nifedipine
also showed some effects on endogenous current of HEK cells,
the average amplitude was about 2 to 3% of the hKv1.5
current. The endogenous current was also less sensitive to
nifedipine, so was unlikely to significantly distort our quantitation of hKv1.5 current block by nifedipine in HEK cells.
The Kd of 6.3 6 0.5 mM for hKv1.5 expressed in HEK cells
was an order of magnitude lower than for an isoform of
hKv1.5 (HPCN1) (Grissmer et al., 1994) expressed in MEL
cells. Possible reasons for this difference are the different
mammalian expression systems (MEL vs. HEK cells) and
small structural differences between the two isoforms of
hKv1.5 used here (fHK vs. HPCN1), although it should be
noted that these isoforms are identical throughout the S4-S6
gating and pore regions. We found that photoinactivation of
the drug occurred readily in the cloned cell system and when
making measurements with nifedipine it was necessary to
carry out all experiments in the dark. A Kd in the low micromolar range, and threshold effects in the 100 nM range make
nifedipine a potent blocker of hKv1.5. No studies have addressed block of specific components of cardiac K1 current in
intact human myocytes by nifedipine, so at the present time
it is not possible to relate our observations directly to currents in human heart.
Time dependence of block and open channel block.
Our data indicated that nifedipine block of hKv1.5 showed
marked time-dependence. Block increased in an exponential
manner during the depolarizing pulses (fig. 2), and the onset
of block occurred sharply after current activation (figs. 3 and
5). Nifedipine also modified the tail current (fig. 6). Upon
repolarization, control channel deactivation was fast and virtually irreversible (fig. 6A). Open-channel models predict
that if a large fraction of the channels is blocked at the start
of repolarization and the unbinding rate (k21) is fast enough,
then the tail may display a rising phase reflecting the unblocking from blocked to open state. Subsequently, the tail
should deactivate more slowly than in control, because some
unblocked channels become blocked again, depending on the
relative rate constants for the open to blocked state and the
open to closed state. Current traces in figure 6C (lower panel)
show that a rising phase was prominent with 10 and 5 mM,
but less so with 2 mM nifedipine and absent in control.
Subsequently tail currents cross-over as channels in the
presence of nifedipine move more slowly from the blocked
state to open and closed states than in control (fig. 6, B and
C, upper panel) (Snyders et al., 1992; Fedida, 1997). From the
results discussed above we conclude that nifedipine binds to
the open state of the channel.
The rate of current decay in the control could be fitted to a
single exponential function, and in the presence of nifedipine,
the inactivation became biexponential. The nifedipine-induced extra component of inactivation had a time constant
that was much faster than that of slow inactivation; therefore, this fast time constant (t2) can be considered to represent the interaction of nifedipine with the open state, t2 5
1/(k11 [D] 1 k21), as described before (Snyders et al., 1992;
Slawsky and Castle, 1994). Based on this interaction, the
apparent association and apparent dissociation rate constants for nifedipine obtained from figure 4B were k11 5
5.64 3 106 M21 sec21 and k21 5 37.5 sec21, respectively. The
resultant Kd value from equation [2b] (see “Materials and
Methods”) was 6.65 mM, which is similar to the Kd value from
Nifedipine Block of hKv1.5
1256
Zhang et al.
11
References
BAUKROWITZ, T. AND YELLEN, G.: Use-dependent blockers and exit rate of the last
ion from the multi-ion pore of a K1 channel. Science 271: 653–656, 1996.
BEZANILLA, F., PEROZO, E., PAPAZIAN, D. M. AND STEFANI, E.: Molecular basis of
gating charge immobilization in Shaker potassium channels. Science 254:
679–683, 1991.
BOUCHARD, R. A. AND FEDIDA, D.: Closed and open state binding of 4-aminopyridine to the cloned human potassium channel Kv1.5. J. Pharmacol. Exp.
Ther. 275: 864–876, 1995.
CATTERALL, W. A.: Structure and function of voltage-sensitive ion channels.
Science 242: 50–61, 1988.
CHARNET, P., OUADID, H., RICHARD, S. AND NARGEOT, J.: Electrophysiological
analysis of the action of nifedipine and nicardipine on myocardial fibers.
Fundament. Clin. Pharmacol. 1: 413–431, 1987.
CHOQUET, D. AND KORN, H.: Mechanism of 4-aminopyridine action on voltagegated potassium channels in lymphocytes. J. Gen. Physiol. 99: 217–240,
1992.
DECOURSEY, T. E.: Mechanism of K1 channel block by verapamil and related
compounds in rat alveolar epithelial cells. J. Gen. Physiol. 106: 745–779,
1995.
FEDIDA, D., WIBLE, B., WANG, Z., FERMINI, B., FAUST, F., NATTEL, S. AND BROWN,
A. M.: Identity of a novel delayed rectifier current from human heart with a
cloned K1 channel current. Circ. Res. 73: 210–216, 1993.
FEDIDA, D.: Gating charge and ionic currents associated with quinidine block of
hKv1.5. J. Physiol. 499.3: 661–675, 1997.
GOTOH, Y., IMAIZUMI, Y., WATANABE, M., SHIBATA, E. F., CLARK, R. B. AND GILES,
W. R.: Inhibition of transient outward K1 current by DHP Ca21 antagonists
and agonists in rabbit cardiac myocytes. Am. J. Physiol. Heart Circ. Physiol.
260: H1737–H1742, 1991.
GRISSMER, S., NGUYEN, A. N., AIYAR, J., HANSON, D. C., MATHER, R. J., GUTMAN,
G. A., KARMILOWICZ, M. J., AUPERIN, D. D. AND CHANDY, K. G.: Pharmacological
characterization of five cloned voltage-gated K1 channels, types Kv1.1, 1.2,
1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol.
45: 1227–1234, 1994.
HAMILL, O. P., MARTY, A., NEHER, E., SAKMANN, B. AND SIGWORTH, F. J.: Improved
patch-clamp techniques for high-resolution current recording from cells and
cell-free membrane patches. Pflugers Arch. 391: 85–100, 1981.
HUME, J. R.: Comparative interactions of organic Ca11 channel antagonists
with myocardial Ca11 and K1 channels. J. Pharmacol. Exp. Ther. 234:
134–140, 1985.
JACOBS, E. R. AND DECOURSEY, T. E.: Mechanisms of potassium channel block in
rat alveolar epithelial cells. J. Pharmacol. Exp. Ther. 255: 459–472, 1990.
JAHNEL, U., KLEMM, P. AND NAWRATH, H.: Different mechanisms of the inhibition
of the transient outward current in rat ventricular myocytes. Naunyn
Schmiedeberg Arch. Pharmacol. 349: 87–94, 1994.
JAN, L. Y. AND JAN, Y.: Voltage-sensitive ion channels. Cell 56: 13–25, 1989.
MCCORMACK, K., JOINER, W. J. AND HEINEMANN, S. H.: A characterization of the
activating structural rearrangements in voltage-dependent Shaker K1 channels. Neuron 12: 301–315, 1994.
MECCA, T. E. AND LOVE, S. D.: Comparative cardiovascular actions of clentiazem, diltiazem, verapamil, nifedipine, and nimodipine in isolated rabbit
tissues. J. Cardiovasc. Pharmacol. 20: 678–682, 1992.
NAKAYAMA, K., TAKI, M., STREISSNIG, J., GLOSSMAN, H., CATTERALL, W. A. AND
KANAOKA, Y.: Identification of 1,4 dihydropyridine binding regions within the
a1 subunit of skeletal muscle Ca21 channels by photoaffinity labelling with
diazepine. Proc. Natl. Acad. Sci. USA 88: 9203–9207, 1991.
RAMPE, D., WIBLE, B., FEDIDA, D., DAGE, R. C. AND BROWN, A. M.: Verapamil
blocks a rapidly activating delayed rectifier K1 channel cloned from human
heart. Mol. Pharmacol. 44: 642–648, 1993.
SANGUINETTI, M. C. AND KASS, R. S.: Voltage-dependent block of calcium channel
current in the calf cardiac Purkinje fiber by dihydropyridine calcium channel
antagonists. Circ. Res. 55: 336–348, 1984.
SCHUSTER, A., LACINOVÁ, L., KLUGBAUER, N., ITO, H., BIRNBAUMER, L. AND HOFMANN, F.: The IVS6 segment of the L-type calcium channel is critical for the
action of dihydropyridines and phenylalkylamines. EMBO J. 15: 2365–2370,
1996.
SLAWSKY, M. T. AND CASTLE, N. A.: 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, 1994.
SNYDERS, D. J., KNOTH, K. M., ROBERDS, S. L. AND TAMKUN, M. M.: Time-,
voltage-, and state-dependent block by quinidine of a cloned human cardiac
potassium channel. Mol. Pharmacol. 41: 322–330, 1992.
SNYDERS, D. J. AND YEOLA, S. W.: Determinants of antiarrhythmic drug action
- Electrostatic and hydrophobic components of block of the human cardiac
hKv1.5 channel. Circ. Res. 77: 575–583, 1995.
STEFANI, E., TORO, L., PEROZO, E. AND BEZANILLA, F.: Gating of Shaker K1
channels: I. Ionic and gating currents. Biophys. J. 66: 996–1010, 1994.
STÜHMER, W., CONTI, F., STOCKER, M., PONGS, O. AND HEINEMANN, S. H.: Gating
currents of inactivating and non-inactivating potassium channel expressed
in Xenopus oocytes. Pflugers Arch. 410: 423–429, 1991.
TAMKUN, M. M., KNOTH, K. M., WALBRIDGE, J. A., KROEMER, H., RODEN, D. M. AND
GLOVER, D. H.: Molecular cloning and characterization of two voltage-gated
K1 channel cDNAs from human ventricle. FASEB J. 5: 331–337, 1991.
UEHARA, A. AND HUME, J. R.: Interactions of organic calcium channel antagonists with calcium channels in single frog atrial cells. J. Gen. Physiol. 85:
621–647, 1985.
VAN WAGONER, D. R., KIRIAN, M. AND LAMORGESE, M.: Phenylephrine suppresses
outward K1 currents in rat atrial myocytes. Am. J. Physiol. Heart Circ.
Physiol. 271: H937–H946, 1996.
Send reprint requests to: Dr. David Fedida, Department of Physiology,
Botterell Hall, Queen’s University, Kingston, Ontario, Canada, K7L 3N6.
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 16, 2017
binding site in Ca
channels (Nakayama et al., 1991;
Schuster et al., 1996), where it is thought that the dihydropyridines block the channel from the extracellular side and
mutations in repeat IVS6 affect binding (Schuster et al.,
1996). The second line of evidence is based on the gating
current measurements (fig. 8). At concentrations required to
produce approximate 50% block of ionic current, nifedipine
had only very small effects on hKv1.5 gating currents. This
suggests a binding site distant from the intracellular mouth
of the pore, where binding of numerous drugs immobilizes
gating charge (Stühmer et al., 1991; Fedida, 1997). At high
concentrations, on-gating currents of hKv1.5 did not change
in the presence of nifedipine compared with the control, but
off-gating currents in the presence of nifedipine were reduced
at voltages positive to 110 mV (fig. 8F), in the voltage range
that channels open. This also supports the idea that nifedipine can bind only to channels in the open state, i.e., at some
site exposed when the pore opens.
If we speculate that nifedipine acts at the external mouth
of open K1 channels as suggested above, and that binding is
coupled in a 1:1 stoichiometry with a univalent entity (i.e.,
K1 ions) that senses the transmembrane electric field, we
can apply equation 3 to observed block (data in fig. 3), with
z 5 1. The data were well fitted to this model, between 120
and 190 mV, at nifedipine concentrations of 5, 10, 20 and 50
mM. The fractional distance, d, was calculated to be between
0.12 and 0.16. This suggested that nifedipine binding may be
coupled to a charged process that sensed '15% of the transmembrane electric field, from the outside, at its binding site.
If a higher valence for the coupling entity was assumed (i.e.,
z 5 2 or 3), d was reduced proportionally, so the value of 0.12
to 0.16 gives an upper limit for the distance into the field.
Further experiments, perhaps utilizing mutated forms of Kv
channels, are required to understand the apparent voltagedependence of nifedipine block of K1 channels, both in native
systems (Jacobs and DeCoursey, 1990) and in our cloned
channel system.
Vol. 281