© 1995 American Heart Association, Inc.
Articles |
Determinants of Antiarrhythmic Drug Action
Electrostatic and Hydrophobic Components of Block of the Human Cardiac hKv1.5 Channel
From the Departments of Medicine and Pharmacology, Vanderbilt University School of Medicine, Nashville, Tenn.
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Abstract |
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Abstract The molecular basis of antiarrhythmic drug action is still poorly understood. We recently reported that block of the human cardiac hKv1.5 channel by quinidine displayed similarity with internal quaternary ammonium block of squid and Shaker potassium channels. To gain further insight into the molecular determinants of the affinity and the stereoselectivity of antiarrhythmic drug action, we studied the effects of quinine (a diastereomer of quinidine), clofilium (a quaternary ammonium class III agent), and tetrapentylammonium (TPeA, a biophysical reference probe for the internal quaternary ammonium binding site). For all compounds, block was voltage dependent, with a steep increase over the voltage range of channel opening and a superimposed weaker voltage dependence at more positive potentials. The latter electrostatic component was similar for all drugs, consistent with a binding reaction sensing
20%
of the transmembrane electrical field. Clofilium and TPeA displayed a
higher apparent affinity (0.15 and 0.28 µmol/L, respectively), and
quinine displayed a lower one (21 µmol/L) compared with quinidine
(6.2 µmol/L). Block development upon depolarization was time
dependent for clofilium and TPeA but slow compared with quinidine. A
time-dependent component was difficult to resolve for quinine, but the
time course of deactivating tail currents was slower than in the
control condition. The resulting crossover phenomenon was also observed
for the quaternary drugs. Compared with TPeA alone, the combined
application of quinine and TPeA resulted in a reduced current that
decayed slower, consistent with competition. When a bimolecular
model for open-channel block was used, the apparent association rate
constants for these drugs were found to be similar [range, 4.5 to 7.7
(µmol/L)-1 · s-1]. The apparent
dissociation rate constants for clofilium (1.9 s-1) and
TPeA (3.6 s-1) were smaller compared with quinidine (34
s-1), whereas that for quinine was faster (
125
s-1). This large range in dissociation rate constants
could explain the differences between these drugs both in kinetics and
affinity. The results are consistent with a general model in
which these agents act as cationic open-channel blockers but with an
affinity largely determined by the intrinsic stability of the
drug-receptor complex. Hydrophobic interactions are most likely
involved in this stabilization of binding.
Key Words: K+ channels • antiarrhythmia agents • quaternary ammonium • quinidine • clofilium
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Introduction |
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Potassium channels regulate excitability of cardiac cells at various levels. They contribute to maintenance of the resting potential, early repolarization, control of action potential duration, and pacemaking activity.1 Prolongation of action potential duration and cardiac refractoriness represents one modality of antiarrhythmic action (class III action).2 Therefore, potassium channels that open in the voltage range of the plateau are potential molecular targets of class III antiarrhythmic agents. Drug-channel interactions have been studied in myocytes isolated from various animal species and, more recently, from human hearts. A major problem in the investigation of basic mechanisms of drug-channel interaction is posed by the need to eliminate multiple overlapping ionic conductances. Heterologous expression of cloned channels can be used to circumvent this problem: the human cardiac potassium channel hKv1.5 can be stably expressed in L cells, from which the current can be recorded without contamination from other voltage-gated currents.3 The properties of hKv1.5 include activation positive to -30 mV, fast activation kinetics, slow and incomplete inactivation, outward rectification, and high sensitivity to 4-aminopyridine.3 Recently, functionally similar currents have been identified in rat and canine myocytes4 5 6 and in human atrial myocytes.7 The properties of the human atrial current are highly similar to those obtained for hKv1.5 in the heterologous expression system.3 7 Therefore, the expression system provides an important tool for the study of functional and pharmacological characteristics of (human) cardiac ion channels.
We have exploited its advantages to demonstrate that quinidine, one of
the most widely used antiarrhythmic agents in the United States, blocks
the hKv1.5 channel at clinically relevant concentrations.8
The results indicated that quinidine acts in its cationic form as an
open-channel blocker, binding in the internal mouth of the channel and
sensing 20% of the electrical field. This mechanism of block
displayed similarity to the block of other delayed rectifier potassium
channels by quaternary ammonium derivatives.9 10 To gain
more insight into the determinants of drug binding in the bimolecular
drug-channel interaction, we now report on the effects of
tetrapentylammonium (TPeA), clofilium (a quaternary ammonium class III
agent),11 and quinine (a diastereomer of quinidine).
We tested for state, time, and voltage dependence of drug action. The
data suggest a general model in which these agents act as cationic
open-channel blockers but with the affinity and kinetics largely
determined by the intrinsic stability of the drug-channel receptor
complex.
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Materials and Methods |
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Transfection and Cell Culture
We used the stable cell line expressing hKv1.5 that has been described in detail elsewhere.3 Cells were cultured in DMEM supplemented with 10% horse serum and 0.25 mg/mL G418. The cultures were passed every 4 to 5 days by using a brief trypsin treatment. Before experimental use, subconfluent cultures were incubated with 2 µmol/L dexamethasone for 24 hours to induce efficient channel expression. The cells were removed from the dish with a rubber policeman, stored at room temperature, and used within 12 hours.
Electrical Recording
Recordings were made with an Axopatch-1A or an
Axopatch-200A patch-clamp amplifier (Axon Instruments) by using the
whole-cell configuration of the patch-clamp technique.12
Micropipettes were pulled from Starbore borosilicate glass (Radnoti
Glass Co) and were heat-polished. Currents were recorded at room
temperature (21°C to 23°C) and were sampled at 2 to 10 kHz after
anti-alias filtering at 1 to 5 kHz. Data acquisition and command
potentials were controlled by a versatile custom-made programmable
stimulator. To ensure voltage-clamp quality, electrode resistance was
kept at <3 M
; the average resistance was 2.1±0.2 M (n=28).
Junction potentials were zeroed with the electrode in the standard bath
solution. Gigaohm seal formation was achieved by suction (19±3 G;
range, 6 to 50 G). After establishing the whole-cell configuration,
the capacitive transients elicited by symmetrical 10-mV voltage-clamp
steps from -80 mV were recorded at 50 kHz (filtered at 10 kHz) for
calculation of capacitive surface area, access resistance, and input
impedance. The average access resistance was 3.7±0.2 M, and after
compensation, the residual access resistance was 1.1±0.2 M. With an
average current of 2.1±0.2 nA at +60 mV (ie, largest currents
associated with strongest depolarization), voltage errors, based on the
calculated residual access resistance, were <2.5 mV.
Solutions and Chemicals
The "intracellular" pipette filling solution contained
(mmol/L) KCl 110, HEPES 10, K4BAPTA 5, K2ATP 5,
and MgCl2 1 and was adjusted to pH 7.2 with KOH, yielding a
final intracellular potassium concentration of 145 mmol/L. The bath
solution contained (mmol/L) NaCl 130, KCl 4, CaCl2 1.8,
MgCl2 1, HEPES 10, and glucose 10 and was adjusted to pH
7.35 with NaOH. Quinidine (quinidine gluconate) was obtained from
Eli-Lilly. Clofilium was a gift from Eli-Lilly. The lipid solubility
(logP) of clofilium (based on octanol:phosphate buffer pH 7.4
partitioning) is 0.75. All other chemical compounds were obtained from
Sigma Chemical Co. All drugs were added from aqueous stock
solutions.
Pulse Protocols and Analysis
The holding potential was -80 mV unless indicated otherwise.
After obtaining control data, bath perfusion was switched to
drug-containing solution. Effects of drug infusion or removal were
monitored with test pulses from -80 to +50 mV, applied every 20
seconds until steady state was obtained. The cycle time for other
protocols was 10 seconds or slower with quinidine and quinine. In the
presence of clofilium and TPeA, the cycle time was slowed to 20 seconds
because of their slow kinetics.
The standard protocol to obtain current-voltage relations and activation curves consisted of 250-millisecond pulses that were imposed in 10-mV increments between -80 and +60 mV, with additional interpolated pulses to yield 5-mV increments between -30 and +10 mV (activation range of hKv1.5). The "steady state" current-voltage relations were obtained by measuring the current at the end of the 250-millisecond depolarizations. Longer pulses (500 to 750 milliseconds) were used with the lower concentrations of TPeA and clofilium, which induced block slowly (see "Results"). For voltages between -80 and -40 mV, we only observed passive linear leak; least-squares fits to these data were used for leak correction. Capacitive transients were subtracted by using an appropriately scaled average of 16 to 32 hyperpolarizing pulses from -80 to -100 mV. Deactivating tail currents were recorded at -30 or -50 mV. The activation curve was obtained from the tail-current amplitude immediately after the capacitive transient. For steady state curves, raw data points were averaged over a small time window (2 to 5 milliseconds). Raw tracings shown for the present study were corrected for linear leak and capacitive transients as described above and digitally filtered at 1 to 2 kHz in the frequency domain after Fourier transformation.
The time courses of tail currents, slow inactivation, and drug-induced
kinetic changes were fitted with a sum of exponentials. The
curve-fitting procedure used a nonlinear least-squares (Gauss-Newton)
algorithm; results were displayed in a linear and semilogarithmic
format together with a plot of the residual deviations of the data from
the fitted curve (difference plot). Goodness of the fit and required
number of exponential components were judged by comparing
2 values statistically (F test) and by inspection
for systematic nonrandom trends in the difference plot.
Analysis of Drug Action
The amount of block was determined as follows: leak-corrected
current in the presence of drug (Idrug) was normalized to
matching control current (Icontrol) to yield fractional
block (f=1-Idrug/Icontrol). Using a
first-order blocking scheme to describe the drug-channel interaction,
we obtained apparent affinity Kd and Hill
coefficient n from fitting the fractional block f at various drug
concentrations ([D])
with (1) f=1/{1+(Kd/[D])n}
The apparent rate constants for binding (k)
and unbinding (l) were obtained from fits
to (2) 
=kx[D]+l
in which is the inverse of the time
constant 
B of block development. A problem
with this linearization is potential inappropriate weighting of the
data. Therefore, we fitted both the linear equation (Equation 2) and
the hyperbolic function
B=1/(kx[D]+l) to the data. Data
are shown in the linear format.
To analyze the voltage dependence of block, we determined
fractional block f at each voltage above -30 mV. Using data points in
the range of full channel opening (see "Results"), the voltage
dependence of block was fitted with the Woodhull
formalism13 : (3) f=[D]/{[D]+Kd*xexp(-
zFE/RT)}
where F is the Faraday constant, R is the gas constant,
E is the imposed voltage, T is the absolute temperature, and z is the
valence (+1 for quaternary ammoniums and cationic quinidine or
quinine). The parameter represents the
fractional electrical distance, ie, the fraction of the transmembrane
electrical field sensed by a single charge at the receptor site.
Kd* represents the affinity at the
reference voltage (0 mV). This represents a direct approach to
analysis of the voltage dependence of block rather than the
indirect approach, in which the same data (normalized block) are
converted into an apparent affinity, which is subsequently fitted with
the exponential
Kd(E)=Kd*xexp(-zFE/RT).
Mathematical Modeling and Statistics
The activation time course of hKv1.5 is sigmoidal3
and presumably requires activation of four subunits. Activation of
Shaker-related channels is a complicated process, details of
which require mathematical models with up to 16 kinetic
states.14 15 In addition, the hKv1.5 channel displays slow
inactivation with two or three exponential components.3
For the purpose of modeling drug-channel interactions, we simplified
the kinetic model (see diagram in Fig 8
) as follows: (1) channel
opening was described by independent activation of four subunits, and
(2) slow inactivation was omitted. To reproduce the time course of
channel opening and tail current deactivation, rate constants for
subunit activation (
) and deactivation (ß) were set to 400 and 1
s-1 (at +50 mV), respectively, and 0.1 and 7
s-1 at -40 mV, respectively. This model is similar to
that used previously to describe the interaction of quinidine with
hKv1.5.8 For these simulations, we used the fourth-order
Runge-Kutta ordinary differential equation solver of the
MATLAB software (The MathWorks Inc).
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Results are expressed as mean±SEM. ANOVA with appropriate post hoc comparisons was used to compare the differences in mean values; a value of P<.05 was considered significant.
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Results |
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Clofilium, TPeA, and Quinine Inhibit hKv1.5 Currents
Fig 1
illustrates the effects of TPeA, clofilium,
and quinine on hKv1.5 currents expressed in L cells. All compounds were
effective when added to the bath superfusion solution, but 20 minutes
was required to achieve steady state effects with TPeA and clofilium.
This was an order of magnitude slower than the time needed for complete
exchange of extracellular solution (<2 minutes). The effects of
quinidine and quinine reached steady state more quickly (<10 minutes)
and were reversible upon washout. With TPeA and clofilium, only partial
reversal was observed upon washout (30% recovery in 30
minutes).
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TPeA was used as a reference probe for the putative internal quaternary
ammonium site9 10 because it should be more membrane
permeant than tetraethylammonium (TEA) on
the basis of its 100-fold higher lipid solubility.16 After
application of TPeA (Fig 1A
through 1C), the current upon
depolarization initially rose as in the control condition. Upon
reaching a slightly reduced peak current level, the hKv1.5 current
declined in a time-dependent manner to a lower steady state level. This
relaxation process superficially resembled channel inactivation but can
most easily be explained by a shift toward a higher binding affinity,
resulting in a time-dependent relaxation to the new equilibrium between
blocked and drug-free channels. Consistent with the high level
of block induced during the depolarizing pulse, the tail currents were
largely suppressed compared with the control condition. For the
quaternary ammonium class III antiarrhythmic agent clofilium, a similar
pattern was observed, as shown in Fig 1D through 1F. A marked
time-dependent relaxation to a high level of block was observed after
equilibration with 300 nmol/L clofilium (Fig 1E), with more extensive
block at 3 µmol/L clofilium (Fig 1F).
To determine possible stereoselectivity in hKv1.5 block, we tested
whether quinine, a diastereomer of quinidine, interacted with this
channel (Fig 1H through 1I). Quinine clearly suppressed the hKv1.5
current in a concentration-dependent manner. However, in contrast to
TPeA and clofilium, a time-dependent relaxation was not readily
resolved, despite a reduction of hKv1.5 current by 50% with 20
µmol/L quinine.
Concentration Dependence of hKv1.5 Inhibition
Fig 2 shows the concentration dependence of hKv1.5
inhibition. For most concentrations, the suppression of hKv1.5 current
after 250 milliseconds was used as an index of block. However, at the
lowest concentrations of TPeA and clofilium, the interaction with
hKv1.5 was too slow to reach steady state within this time window. In
that case, the suppression after 500 or 750 milliseconds was compared
with the control condition. The dashed line in Fig 2 illustrates the
concentration dependence of hKv1.5 block by quinidine, with an apparent
Kd of 6.2 µmol/L as described
previously.8 Both TPeA and clofilium were more potent than
quinidine, with apparent affinities of 280 and 150 nmol/L,
respectively. In contrast, the apparent affinity for quinine was
significantly less (21 µmol/L). The concentration dependence for
hKv1.5 inhibition was well approximated with a Hill coefficient of 1,
consistent with a bimolecular reaction as was previously
reported for quinidine.8 Admittedly, this could not be
rigorously assessed for clofilium because of the inability to separate
slow and limited block at low concentrations (<300 nmol/L) from the
normal slow and partial inactivation of hKv1.5. To limit the latter, we
attempted to quantify the level of block by using pulse trains with
250-millisecond depolarizations. However, even at 1 µmol/L this
usually resulted in somewhat lower levels of block, which were
presumably due to unblocking between depolarizations.
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Voltage Dependence of Drug-Channel Interactions
Current-voltage relations for quasi–steady state current at the
end of voltage steps, as in Fig 1, indicated that each drug reduced
hKv1.5 current over a wide voltage range. To quantify the effects of
voltage on the drug-channel interaction, we determined fractional block
from the relative suppression of current at each voltage (see
"Materials and Methods"). Fig 3 shows typical
examples and illustrates that the voltage dependence displayed a
similar pattern for all three drugs. A steep phase was visible between
-30 and -10 mV, which coincides with the steep voltage dependence of
channel opening. An additional weaker voltage dependence was observed
in the voltage range positive to 0 mV, over which the hKv1.5 channel is
always open.3 The steepness of this shallow phase was
e-fold per 120 to 150 mV. This voltage dependence can be
explained if the positively charged nitrogen of TPeA and clofilium
senses a fraction of the transmembrane electrical field at the binding
site. When a single barrier model (Equation 3>, see "Materials and
Methods") was used, the calculated fractional electrical distance
was 0.19 and 0.15 for the examples shown in Fig 3, panels A and B,
respectively. In the case of quinine (Fig 3C), a similar calculation
yielded an equivalent electrical distance of 0.18, consistent
with the predominantly cationic form of this drug at
physiological pH (pKa=8.6 to 8.9). The
Table summarizes the average values (n=4 to 6) for the
electrical binding distance obtained in this manner. The values were
close to 0.20, and no significant differences were observed between
these drugs.
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, steep voltage dependence of block coinciding
with channel activation; and 
, shallow voltage dependence
of block. Only the latter were used in the fit with the Woodhull model
(Equation 3>; see "Materials and Methods"), shown by the solid
line with the indicated values for the equivalent electrical distance
(
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Time Dependence of Drug-Channel Interactions
The results presented thus far dealt with steady state
levels of inhibition. In the presence of clofilium or TPeA, a clear
time-dependent decline of the current was observed after channel
opening (Fig 1). If these drugs have a low affinity for the rested
state of the channel but a high affinity for the open conformation, as
suggested by the results in the previous sections, then this time
dependence would represent the time course of relaxation toward
a new equilibrium. The superimposed tracings at +60 mV (insets in Fig 4) illustrate that the rate of this relaxation was
enhanced by increasing the drug concentration, consistent with
the interpretation of this decay as the time course of open-channel
block. This time-dependent block was observed over the entire voltage
range at the higher concentrations for clofilium and TPeA (Fig 1C and 1F). In contrast, the tracings at -20 mV and -10 mV in 0.3 µmol/L
clofilium (Fig 1E) did not display the same extensive time-dependent
decline as the corresponding tracing at +50 mV. Thus, although the time
dependence was obvious at voltages at which the channel
activates quickly, it was apparently obscured by the slower
activation at modest depolarizations. Therefore, we determined the rate
of block from the inverse of the time constant of the drug-induced
exponential component in the hKv1.5 time course at +50 and +60 mV.
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For a bimolecular reaction, the rate of block is a linear function of
the drug concentration (Equation 2). This was the case for both TPeA
and clofilium, as shown in Fig 4, in which the solid line
represents the linear least-squares fit to the data. From the
latter, we obtained apparent rate constants for drug binding
(k) and unbinding (l). Values and
statistics are summarized in the Table, which for comparison also lists
the values for quinidine obtained previously.8 Whereas the
association rate constants differed less than twofold, the unbinding
rate constants for clofilium and TPeA were an order of magnitude slower
than that for quinidine.
Stereoselective Difference in Block by Quinidine and
Quinine
Fig 5 compares the effects of quinidine and
quinine. The kinetic difference between both diastereomers is
especially evident when comparing the tracings for 6 µmol/L quinidine
and 20 µmol/L quinine, which induced comparable levels of block. With
quinidine a time-dependent component of hKv1.5 block was evident (Fig 5A), as has been described in detail before.8 With quinine
it was difficult to identify a time-dependent component of block after
channel opening (although we occasionally observed a small and brief
decaying transient). Nevertheless, the tracings in the presence of
quinine were not a scaled-down version of the control current. Instead,
the transition from the rising phase into the sustained current
appeared more abrupt. As shown by the modeling (see
"Discussion"), this is consistent with a fast interaction
with the open state. To further illustrate the reversibility and the
stereoselective difference between quinine and quinidine, Fig 5C shows
the current-voltage relations for an experiment in which both drugs
were applied in the same cell. Application of 20 µmol/L quinine
induced 48% block at +50 mV. After a 20-minute washout, a virtually
complete reversal of this effect was obtained. Subsequently, an
equimolar concentration of quinidine was applied, inducing
substantially more block (77%). Both levels of block were
consistent with the apparent affinities from Fig 2.
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Modification of Tail Current Time Course
If these drugs only interact with the open hKv1.5 channel, then
blocked channels need to transit through the open state before they
could deactivate to the rested state (see diagram in Fig 8). Thus, one would expect a modification of the deactivating tail
currents. Fig 6 shows tail currents in the control
condition and after TPeA, clofilium, and quinine in concentrations that
resulted in 30% to 60% block during the eliciting depolarization.
Compared with the control condition, the tail currents in each case
started at a lower amplitude, reflecting the depolarization-induced
block. The subsequent decay was markedly slower than in the control
condition, and superposition of tail currents in the control condition
and in the presence of drug resulted in a "crossover" phenomenon
(arrows in Fig 6).
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Effects Combined Application of TPeA and Quinine
To further determine whether quinine binds at the same (or an
overlapping) site, we compared the effect of combined application of
quinine and TPeA with TPeA alone. In these experiments, the cells were
preincubated with TPeA for at least 30 minutes. If both drugs bind
independently (ie, at different sites), then the addition of quinine
would further reduce the current. Fig 7A shows that this
was not the case. In the presence of both drugs, the peak current was
suppressed, but the hKv1.5 current decayed much slower despite the
higher total concentration of blockers. This resulted in a larger
amount of current later during depolarization. Similar observations
were made in five additional experiments. With sufficient separation in
interaction rates, the relative blocking rate should be proportional to
the relative suppression of
current17 18 : (4) TQ/T=ITQ/IT
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In this relation, T and TQ
are the apparent blocking rates for TPeA and the TPeA/quinine
combination, respectively; ITQ/IT is the
fraction of remaining peak current in the TPeA/quinine combination
versus TPeA. In four experiments with 20 µmol/L quinine, the relative
peak amplitude averaged 0.68±0.03, and the relative blocking rate
averaged 0.63±0.07 (n=4, P=NS). The above relation does not
depend explicitly on the drug concentration and should be satisfied
when the quinine concentration is changed in the continued presence of
TPeA. This is illustrated for three experiments in Fig 7B. In each
case, this proportionality was observed during administration or
washout of quinine, as indicated by the clustering of the data around
the predicted line.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main observations in the present study were as follows: (1) Block of hKv1.5 by tertiary amine drugs quinidine and quinine resembled that by quaternary ammonium drugs in state, time, and voltage dependence, pointing to a common mechanism (open-channel block). (2) Stereoselective differences exist between quinidine and quinine in affinity and kinetics of hKv1.5 block. (3) The high-affinity blockers displayed slow kinetics of block. (4) Quinine and TPeA displayed interaction, suggesting competition for a common site. (5) The difference in kinetics and affinity could be explained largely by the 100-fold range in the rate constants for drug dissociation from the channel receptor.
Block of the Open Conformation of hKv1.5
TPeA and clofilium both reduced the open-channel current (Figs 1 and 4). Upon depolarization, the channel started to open as in the
control condition and reached a peak current level that was only
slightly depressed compared with the control condition (Fig 1B and 1E).
This indicates that little or no channel block existed at -80 mV and
that the kinetics of block were too slow to produce a significant level
of inhibition before reaching the peak current level. The difference
between absence of block at -80 mV and the high steady-state level of
block upon depolarization is not consistent with a
state-independent model for block. It indicates that channel opening is
required before binding can occur. In this case, block is expected to
track the voltage dependence of channel opening, explaining the steep
phase of the voltage dependence of block in Fig 3.
Development of block by TPeA and clofilium was considerably slower than
that produced by quinidine.8 The rate of block increased
linearly with drug concentration (Fig 4), as expected for open-channel
block. The derived association rate constant k for both
drugs was similar to that for quinidine (Table). The main difference
with quinidine was a 10-fold slower apparent unbinding rate for both
drugs. These rate constants can also be used to estimate the apparent
Kd (=l/k), yielding values of 0.8 and
0.25 µmol/L for TPeA and clofilium, respectively. This estimate is
independent from the apparent Kd obtained from
steady state current suppression (Fig 2, Table). Nevertheless, the
values are in reasonable agreement, and the small differences probably
result from the interference of the intrinsic slow inactivation of
hKv1.5 with both measurements.
Fig 8A shows that mathematical simulations using an
open-channel block model reproduced the essential features of the
interaction between clofilium and hKv1.5, using the rate constants from
the Table. With increasing drug concentration, both the extent of block
and the rate of its development increased, similar to the experimental
results (Figs 1 and 4B). Fig 8A shows that the pure open-channel block
model can also account for a concentration-dependent reduction in the
peak outward current upon depolarization, as consistently seen
in the experimental tracings (Figs 1 and 4).
Fast Open-Channel Block Can Explain the Effects of
Quinine
The lack of a clear time-dependent component of block after
channel opening might suggest a different mechanism of action for
quinine. However, observations supporting open-channel block include
the biphasic voltage dependence of block similar to that for the
quaternary compounds (Fig 3) and quinidine,8 the similar
binding distance, and the tail current crossover (Fig 6C). Therefore,
we tested whether a kinetic change in the open-channel block model was
sufficient to account for the interaction of quinine with hKv1.5.
Lacking a direct experimental measure of the binding rate, we reasoned
as follows: If the more abrupt transition (compared with control) from
the rising phase of channel opening into the reduced steady state level
represents fast block, ie, block occurring on the time scale of
channel opening, then the time constant for 20 µmol/L quinine should
be <4 to 5 milliseconds. At the Kd, the
forward rate kx[D] equals the off rate l, or
the binding rate (=kx[D]+l) equals
2l. With the constraint that the time constant should be <4
milliseconds, we would expect 2l to be >250
s-1 or l to be >125 s-1. Taking
the latter lower limit of 125 s-1 for l, the
association rate constant would be
k=6 · s-1, ie, in the range of the
binding rate constants for quinidine and the quaternary ammonium
derivatives. Fig 8B shows the modeled-kinetics change in function of
the dissociation rate constant for three hypothetical drugs that have
the same association rate constant (k) and were applied in a
concentration ([D]) corresponding to their Kd.
The cases for l=2, 30, and 120 s-1 correspond
roughly to clofilium, quinidine, and quinine, respectively. With an
increase in the dissociation rate constant, the phasic component became
faster and was reduced in amplitude. For l=120
s-1, the current was suppressed almost immediately,
leaving only a small fast phasic component. In other simulations with
l=200 s-1 and k=9.7
(µmol/L)-1 · s-1
(Kd=20.6 µmol/L), the phasic component was
further reduced to <3% of the total amplitude (not shown). In actual
experiments, it is not surprising that such small components cannot be
resolved reliably and essentially appear as a sharp transition from the
rising phase of the current into the blocked state. Thus, the lack of
clearly resolved time dependence does not require the postulation of a
separate mechanism for hKv1.5 block by quinine.
The data illustrated in Fig 7 further address site and kinetics of
quinine block. These results resemble the competitive interaction of
internal TEA with N-type ("ball and chain")
inactivation.17 18 With independent binding, the current
in the presence of both drugs would be scaled down compared with TPeA
alone. However, if the return from the TPeA blocked state is slow and
if quinine interacts much faster, then the competitive scheme predicts
that the apparent blocking rate is reduced proportionally to the
depression of the peak current.17 18 This competition is
further illustrated by the larger current (crossover) later in the
step. This paradoxical increase in current is clearly
inconsistent with independent binding but a logical
consequence of the competition: channels blocked by either compound are
nonconducting, but in the presence of quinine, the channels shuttle
frequently between the short-lived blocked state and the open state.
Each time the channel opens the probability to become blocked again by
quinine exceeds that of becoming "irreversibly" blocked by TPeA.
Thus, these data further demonstrate that quinine interacts with hKv1.5
significantly faster than does TPeA but binds at the same or a nearby
site.
Electrostatic Effect of the Transmembrane Electrical Field on
Drug Binding
As shown in Fig 3, a shallow voltage dependence of binding was
observed positive to 0 mV, which is the voltage range over which the
hKv1.5 channel is fully open.3 In terms of drug-channel
interaction, this effect could result from properties of the drug or
the receptor. It is conceivable that the affinity of the open-channel
receptor displays itself an intrinsic and continuous voltage
dependence. As argued elsewhere,8 this is unlikely, and a
simpler explanation is that these charged drugs bind at a receptor site
within the transmembrane electrical field. The electrical distance
calculated from the Woodhull model (Snyders et al8 ) was
0.19 for quinidine, and comparable values were obtained for the drugs
in the present study (Fig 3, Table). This indicates binding at a
site 20% into the transmembrane electrical field as referenced from
the intracellular side. The similarity in this binding distance and the
similarity in the overall mechanism of hKv1.5 inhibition strongly
suggest that all these drugs bind either at the same receptor or at
least that when bound to their receptors, the positively charged amine
is approximately in the same position.
In Shaker channels, residues at the putative innermost turn
of the P loop and in the carboxyl terminal half of S6 have been
implicated in the binding of quaternary ammonium derivatives, including
clofilium.10 19 The electrical binding distance obtained
for the drugs in the present study is similar to the value observed
for internal TEA block of Shaker channels.10 20
The affinity observed for TPeA in this human Shaker-like
channel is very similar to the affinity of the longer chain
N-alkyltriethylammonium derivatives in the Shaker
channel.10 Both affinity and interaction rate constants
for TPeA are also very similar to those obtained for
Kv1.6.21 Our results with clofilium are in agreement with
those obtained by Malayev et al22 in terms of affinity,
kinetics during depolarization, and overall interpretation as
open-channel block from the inside. The slow dissociation rate (Table)
may correspond to the fast component of recovery from block in their
study.
The shallow voltage dependence observed positive to 0 mV for each of the drugs in the present study is similar to that obtained for other blockers of hKv1.5, such as terfenadine,23 24 verapamil,25 and bupivacaine.26 Thus, the electrical field appears to influence drug binding in a similar fashion for each of these drugs, consistent with a common binding site for antiarrhythmic drugs in the internal mouth of the channel. Nevertheless, this electrostatic component may not contribute much to the binding energy, because it is similar for these drugs despite a 100-fold difference in affinity. The main effect of this electrostatic component may be either to facilitate diffusion to the binding site or to help orient the molecule in a favorable way for binding at the receptor.
Hydrophobic Determinant of Block
The second determinant of binding is suggested by the large range
of apparent dissociation rate constants. These did correlate well with
the changes in affinity, which suggests that they reflect a large
fraction of the binding energy (Table). The dissociation rate constant
reflects the stability of the drug-receptor complex. Because the
electrostatic component is similar, as is the charge on these drugs, we
propose that this indicates that hydrophobic interactions are important
in determining the stability of the drug-channel complex. A similar
explanation was proposed for the interaction of various quaternary TEA
derivatives with squid potassium channels9 27 and for the
interaction of TEA analogues with Shaker
channels.10 Such a model could also explain the seemingly
paradoxical result that the affinity for open sodium channel blockers
correlated strongly with their hydrophobicity, as judged from their
logP.28 Thus, these open-channel blockers appear to gain
access to the receptor via a hydrophilic pathway,29 but
hydrophobic interactions with the wall of the internal mouth of the
channel appear to be the major determinant of the affinity.
Interestingly, the polar substitution on the aromatic ring of the
methanesulfonanilide class III drugs seems to be incompatible with the
structure of the binding site in these channels, because no block was
detected with 1 mmol/L D-sotalol30 or 1
µmol/L dofetilide (authors' unpublished observations, 1994).
Our results with the quaternary drugs display similarity with clofilium block of batrachotoxin-modified sodium channels for which hydrophobic interactions were proposed,31 and with block of normal cardiac sodium channels by quarternary drugs.32 33 34 Furthermore, the S6 segment of domain IV of a cloned sodium channel has been implicated in the binding of local anesthetics.35 The similarity may reflect a common tertiary structure of the internal vestibule of these voltage-gated ion channels.
Conclusion
In summary, we propose that binding of several class III
antiarrhythmic drugs to hKv1.5 potassium channels is determined by an
electrostatic component reflected in the electrical binding distance
and a hydrophobic component that determines the affinity and depends on
the nature and orientation (stereoselectivity) of the side chains.
Because of the similarity between the S6 segment of these potassium
channels with the corresponding segment in domain IV of sodium and
calcium channels, this may represent a general set of
determinants for drug binding in the internal mouth of voltage-gated
ion channels. An unresolved question is why these channels have such a
binding site, but one could speculate that these drugs use a structure
that is needed for normal channel function.
|
Acknowledgments |
|---|
This study was supported by National Institutes of Health grants HL-47599 and HL-46681. The authors wish to thank Drs Roden and Tamkun and Mr Rich for review of the manuscript, Dr Bennett for helpful discussions, Mr Short and Mr Kodali for technical help, and Mr Sheffer for secretarial help.
|
Footnotes |
|---|
Reprint requests to Dirk J. Snyders, MD, 554-MRB2, Vanderbilt University School of Medicine, Nashville, TN 37232-6602.
Previously presented as preliminary results in abstract form (Biophys J. 1992;61:151a and Circulation. 1992;86[suppl I]:I-26).
Received July 8, 1994; accepted May 31, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Gintant GA, Cohen IS, Datyner NB, Kline RP. Time-dependent outward currents in the heart. In: Fozzard HA, Haber E, Jennings RB, Katz AM, Morgan HE, eds. The Heart and Cardiovascular System: Scientific Foundations. New York, NY: Raven Press Publishers; 1992:1121-1169.
2. Vaughan-Williams EM. Classification of antiarrhythmic actions. In: Vaughan-Williams EM, ed. Handbook of Experimental Pharmacology. 1989;89:45-62.
3.
Snyders DJ, Tamkun MM, Bennett PB. A rapidly
activating and slowly inactivating potassium channel cloned from human
heart: functional analysis after stable mammalian cell culture
expression. J Gen Physiol. 1993;101:513-543.
4.
Boyle WA, Nerbonne JM. A novel type of
depolarization-activated K+ current in adult rat
atrial myocytes. Am J Physiol. 1991;260:H1236-H1247.
5.
Boyle WA, Nerbonne JM. Two functionally
distinct 4-aminopyridine sensitive outward K+ currents
in rat atrial myocytes. J Gen Physiol. 1992;100:1041-1067.
6.
Jeck CD, Boyden PA. Age-related appearance of
outward currents may contribute to developmental differences in
ventricular repolarization. Circ
Res. 1992;71:1390-1403.
7.
Wang Z, Fermini B, Nattel S. Sustained
depolarization-induced outward current in human atrial myocytes:
evidence for a novel delayed rectifier K+ current similar
to Kv1.5 cloned channel currents. Circ
Res. 1993;73:1061-1076.
8. Snyders DJ, Knoth KM, Roberds SL, Tamkun MM. Time-, voltage-, and state-dependent block by quinidine of a cloned human cardiac potassium channel. Mol Pharmacol. 1992;41:322-330. [Abstract]
9.
Armstrong CM. Interaction of
tetraethylammonium ion derivatives with the
potassium channels of giant axons. J Gen
Physiol. 1971;58:413-437.
10. Choi KL, Mossman C, Aube J, Yellen G. The internal quaternary ammonium receptor site of Shaker potassium channels. Neuron. 1993;10:533-541. [Medline] [Order article via Infotrieve]
11. Arena JP, Kass RS. Block of heart potassium channels by clofilium and its tertiary analogs: relationship between drug structure and type of channel blocked. Mol Pharmacol. 1988;34:60-66. [Abstract]
12. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 1981;391:85-100. [Medline] [Order article via Infotrieve]
13.
Woodhull AM. Ionic blockade of sodium channels
in nerve. J Gen Physiol. 1973;61:687-708.
14. Bezanilla F, Perozo E, Stefani E. Gating of Shaker K+ channels, II: the components of gating currents and a model of channel activation. Biophys J. 1994;66:1011-1021. [Medline] [Order article via Infotrieve]
15.
Zagotta WN, Hoshi T, Aldrich RW.
Shaker potassium channel gating, III: evaluation of
kinetic models for activation. J Gen
Physiol. 1994;103:321-362.
16. Hansch G, Leo A. Substituent Constants for Correlation Analysis in Chemistry and Biology. New York, NY: John Wiley & Sons Inc; 1979.
17.
Choi KL, Aldrich RW, Yellen G.
Tetraethylammonium blockade
distinguishes two inactivation mechanisms in voltage-activated
K+ channels. Proc Natl Acad Sci U S A. 1991;88:5092-5095.
18. Grissmer S, Cahalan M. TEA prevents inactivation while blocking open K+ channels in human T lymphocytes. Biophys J. 1989;55:203-206.[Medline] [Order article via Infotrieve]
19. Yool AJ. Block of the inactivating potassium channel by clofilium and hydroxylamine depends on the sequence of the pore region. Mol Pharmacol. 1994;46:970-976. [Abstract]
20.
Yellen G, Jurman ME, Abramson T, MacKinnon R.
Mutations affecting internal TEA blockade identify the probable
pore-forming region of a K+ channel.
Science. 1991;251:939-942.
21.
Kirsch GE, Taglialatela M, Brown AM. Internal
and external TEA block in single cloned K+
channels. Am J Physiol. 1991;261:C583-C590.
22. Malayev AA, Nelson DJ, Philipson LH. Mechanism of clofilium block of the human Kv1.5 delayed rectifier potassium channel. Mol Pharmacol. 1995;47:198-205.[Abstract]
23. Rampe D, Wible B, Brown AM, Dage RC. Effects of terfenadine and its metabolites on a delayed rectifier K+ channel cloned from human heart. Mol Pharmacol. 1993;44:1240-1245. [Abstract]
24. Yang T, Prakash C, Roden DM, Snyders DJ. Mechanism of block of a human cardiac potassium channel by terfenadine racemate and its enantiomers. Br J Pharmacol. 1995;115:267-274. [Medline] [Order article via Infotrieve]
25. Rampe D, Wible B, Fedida D, Dage RC, Brown AM. Verapamil blocks a rapidly activating delayed rectifier K+ channel cloned from human heart. Mol Pharmacol. 1993;44:642-648. [Abstract]
26. Valenzuela C, Delpon E, Tamkun MM, Tamargo J, Snyders DJ. Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers. Biophys J. 1995;69:418-427. [Medline] [Order article via Infotrieve]
27.
Swenson RP. Inactivation of potassium current in
squid axon by a variety of quaternary ammonium ions.
J Gen Physiol. 1981;77:255-271.
28.
Ehring GR, Moyer JW, Hondeghem LM. Quantitative
structure activity studies of antiarrhythmic properties in a series of
lidocaine and procainamide derivatives. J
Pharmacol Exp Ther. 1988;244:479-492.
29.
Hille B. Local anesthetics: hydrophilic and
hydrophobic pathways for the drug-receptor reaction.
J Gen Physiol. 1977;69:497-515.
30. Grissmer S, Nguyen AN, Aiyar J, Hanson DC, Mather RJ, Gutman GA, Karmilowicz MJ, Auperin DD, Chandy KG. Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol Pharmacol. 1994;45:1227-1234. [Abstract]
31. Wang GK, Simon R, Bell D, Mok WM, Wang S. Structural determinants of quaternary ammonium blockers for batrachotoxin-modified Na+ channels. Mol Pharmacol. 1994;44:667-676. [Abstract]
32.
Gingrich KJ, Beardsley D, Yue DT. Ultra-deep
blockade of Na+ channels by a quaternary ammonium ion:
catalysis by a transition-intermediate state? J
Physiol (Lond). 1993;471:319-341.
33. Nettleton J, Castle NA, Wang GK. Block of single batrachotoxin-activated Na+ channels by clofilium. Mol Pharmacol. 1991;39:352-358. [Abstract]
34.
O'Leary ME, Horn R. Internal block of human
heart sodium channels by symmetrical tetra-alkylammoniums.
J Gen Physiol. 1994;104:507-522.
35.
Ragsdale DS, McPhee JC, Scheuer T, Catterall WA.
Molecular determinants of state-dependent block of
Na+ channels by local anesthetics.
Science. 1994;265:1724-1728.
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