Extract
Note: Please read the complete
full text with Figures and Tables at
Introduction
The human-ether-a-go-go-related gene
(HERG)[1] encodes the pore-forming α-subunits of channels that conduct the rapid delayed rectifier
K+ current
IKr[2].
IKr is one of the most important membrane currents responsible for ventricular action potential repolarization. The suppression of HERG
channels can lead to electrocardiographic changes including action potential and QT interval prolongation, which can be
both antiarrhythmic and cause long-QT syndrome
(LQTS)[3]. More commonly, LQTS is an adverse
effect of many different types of drugs, including antiarrhy-thmics, antihistamines, antibiotics, gastrointestinal prokine-tics,
and antipsychotics[4,5]. It has been documented that drug-induced QT prolongation is mainly due to the drug-mediated
inhibition of IKr, although these drugs are structurally
diverse[4_7].
Structure function data provides evidence that the HERG
channel possesses a larger pore cavity than other
voltage-gated K+ (Kv) channels. Moreover, the aromatic amino
acids which present in the inner (S6) helices of HERG are
absent from Kv channels, which form key components of a
high-affinity drug binding site. These features appear to
confer upon the HERG channel a unique susceptibility to
pharmacological blockade[8].
Because of their potential pro-arrhythmic effects, a
number of non-cardiac drugs have been withdrawn from the
market (eg terfenadine, cisapride, sertindole, grepafloxacin, and
thioridazine) and many have been labeled for restricted use
(eg mesoridazine, ziprasidone, droperidol, astemizol, and
arsenic trioxide). Therefore, screening compounds for HERG
and QT interval liability is now routine in the pharmaceutical
industry. To facilitate the rational design of safer drugs
without HERG liability, it is important to understand the
biophysical and molecular mechanisms of HERG blocked by
drugs. Verapamil, an L-type calcium antagonist, is useful in
the treatment of hypertension, stable angina, and narrow
QRS complex supraventricular arrhythmias. In addition to
blocking L-type Ca2+ channels, verapamil is a potent
antagonist of
IK[9_11]. In a previous study, it was reported that
wild-type (WT) HERG is expressed in human embryonic kidney
(HEK 293) cells with an estimated half-maximal inhibition
concentration (IC50) of 143.0 nmol/L, and the C-type
inactivation-deficient mutations, Ser620Thr and Ser631Ala, lied in
the S5-S6 linker near the internal and the external mouth of
the channel pore, reduced verapamil blockade, which is
consistent with a role for C-type inactivation in high-affinity
drug blockade[10]. At the same time, Lang and colleagues
showed that verapamil and its enantiomers inhibited HERG
K+ channels expressed in Xenopus oocytes with half-
maximal inhibitory concentration (IC50) values of 2.2_4.0
µmol/L[11]. However, until now, the biophysical and
molecular mechanisms of HERG blockade by verapamil have been
unclear. Accordingly, we used HERG expressed in oocytes
to investigate the inhibitory action on HERG
K+ channel current
(IHERG) by verapamil, and determined whether or not
key molecular determinants of HERG blockade for previously
investigated drugs[12] are also important for the inhibition of
IHERG by verapamil.
Materials and methods
Oocyte preparation Oocytes were isolated by
dissection from adult Xenopus laevis. The frogs were
anaesthetized by ice for 20_30 min. After dissection and the removal
of the ovarian lobes, the incision was sutured and the frogs
were allowed to recover for about 1 month before the
removal of a second set of oocytes. Clusters of oocytes were
digested with 1.5 mg/mL Type IA collagenase (Sigma, St Louis, MO,
USA) in a Ca2+-free ND96 solution which contained 96
mmol/L NaCl, 2 mmol/L KCl, 2 mmol/L
MgCl2 , and 5 mmol/L
N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid
(HEPES), pH 7.6 for 1.5 h, and washed extensively with
Ca2+-free solution without collagenase.
cRNA preparation and injection HERG channel site-
directed mutagenesis (Y652A and F656A) were chosen for
the study (Figure 1). Wild-type Y652A and F656A HERG
cDNA were generously donated by Dr Michael
SANGUINETTI (Utah University, Salt Lake City, Utah, USA).
Complementary RNA for injection into oocytes were prepared with the
mMESSAGE mMACHINE kit (Ambion, Austin, TX, USA) after linearization of the expression construct with
EcoR I.
Stage V_VI deflocculated X laevis oocytes were injected
with 50 nL cRNA (1 µg/µL) per oocyte using a Nanoject
microdispenser (Drummond Scientific, Broomhall, PA, USA)
and incubated at 18 °C in ND96 solution (96 mmol/L NaCl, 2
mmol/L KCl, 1.8 mmol/L CaCl2, 1 mmol/L
MgCl2, and 5 mmol/L HEPES, pH 7.6) supplemented with 100 U/mL penicillin.
Electrophysiological experiments Electrophysiological
measurements were performed 2_10 d after oocyte injection.
The 2-microelectrode voltage clamp was used to record
currents from X laevis oocytes. The microelectrodes were filled
with 3 mol/L KCl and had a resistance of 1_5 MΩ.
Recordings were performed using a commercially available amplifier
(Warner OC-725A, Warner Instruments, Hamden, CT, USA)
and pCLAMP software (Axon Instruments, Foster City, CA,
USA) for data acquisition and analyses. Currents were
recorded in an ND 96 bath solution at room temperature (20_
23 °C). The current signals were low-pass-filtered at 500 Hz
and no leak subtraction was used. Oocytes were kept in the
current-clamp mode for at least 5 min before switching to
voltage-clamp mode. Only oocytes exhibiting a resting
potential less than -40 mV were used.
Drugs Verapamil (Sigma, USA) was prepared as a stock
solution (25 mmol/L). Before the experiments, the stock
solution was diluted with bath solution to reach the desired
final concentration. During the experiments, the
cell-superfusate was exchanged using a home-built solution
application device capable of changing the solution bathing
a cell in less than 10 s. In general, recordings were started 60
s after the solution switch. All measurements were performed
under steady-state conditions at least 2 min after the total
solution exchange.
Statistical analysis The digitized data were analyzed
with pCLAMP software (Axon Instruments, USA) and
ORIGIN software (Origin Lab Corporation, Northampton, MA,
USA) and Excel (Microsoft, Redmond, WA, USA). The
concentration-response curves were fitted with a logistic
dose-response equation to obtain the
IC50 values. To determine the voltage dependence of HERG current activation, a least
squares algorithm on ORIGIN software or Excel was used to
fit tail current amplitudes
(Itail) to a Boltzmann equation. The
corrected steady-state inactivation curves were also fitted
with a Boltzmann equation. All values are presented as
mean±SEM. An analysis by Student's t-test was performed
for paired or unpaired observations. A P value of less than
0.05 was considered significant.
Results
Voltage-dependent block of WT and Y652A HERG
channels by verapamil The voltage dependence of
IHERG blockade was determined using the protocol shown in the inset of
Figure 2A. Representative WT and Y652A HERG current
traces in the control and in the presence of verapamil are
respectively shown in Figure 2A, 2B. Both activating
currents measured at the end of the depolarizing step
(Istep) and peak tail current amplitude
(Itail) measured following the step
to -60 mV were dramatically reduced by verapamil (Figure 2).
After the application of 10 µmol/L verapamil, the peak of the
I-V relationship for both channels were shifted to the left
(Figure 2C, 2D), suggesting a negative shift in the voltage
dependence of activation. This was confirmed by a tail
current analysis (Figure 2G, 2H). The current-voltage plot for
tail currents (control Itail and verapamil
Itail at -60 mV) was fitted with a Boltzmann equation to obtain the mid-point
activation voltage (V1/2) and slope factor. For WT HERG
channels, the half-point activation value was -19.73±0.46 mV
(control) and -23.00±0.59 mV with verapamil
(P<0.05, 6 oocytes), with no significant change in slope factors (8.6±
0.28 mV and 8.1±0.53 mV for the control and verapamil,
respectively, P>0.05). For the Y652A HERG channel, the
half-point activation value was -20.29±0.21 mV (control) and
-22.10±0.43 mV with verapamil (P<0.05, 6 oocytes), again with
no significant change in slope factors (7.98±0.12 mV and
7.54±0.32 mV for the control and verapamil, respectively,
P>0.05).
The voltage dependence of WT and the Y652A HERG
tail current block by verapamil are shown respectively in
Figure 2I, 2J, where the relative tail current represents the
ratio of the peak tail currents measured in the presence and
absence of verapamil. The steady-state reduction of the
WT HERG current by 10 µmol/L verapamil varied as a
function of test potential, with the fractional decrease varying
from 0.5 at -50 mV to 0.62 at +20 mV (Figure 2I). To achieve
equivalent blockade, we used 80 µmol/L verapamil for Y652A.
The steady-state block of Y652A HERG was also voltage
dependent and varied from 0.48 at -50 mV to 0.63 at +20 mV
(Figure 2J). Verapamil blocked Y652A HERG channels only
after opening the activation gate. That is consistent with
WT HERG. For both channels, the blockade of the HERG
current by verapamil increased significantly over the
voltage range where HERG channels activated and saturated at
voltages where HERG activation was maximal. These results
are consistent with the open channel blockade.
To investigate the state-dependence of the HERG
channel blockade by verapamil, a single long test pulse from -90
mV to 0 mV (4000 ms) was used. After the control
measure-ment, HERG channels were kept in the closed state at a
potential of -90 mV, and 10 µmol/L verapamil was perfused into
the bath for 10 min to allow for the equilibration of drug
concentrations within the bath and the cell. Then the step
protocol was repeated and recorded (n=6, Figure 3A). By
the division of currents and presentation in a normalized
form, the time course of relative block was obtained (Figure
3B). The fractional block increased rapidly following
membrane depolarization, but reached a plateau after 1 s,
indicating time dependence of inhibition upon depolarization. This
observation argues against closed-channel inhibition and
for a dependence of IHERG blockade on channel gating.
Inactivation of WT and Y652A HERG channels by
verapamil The effect of verapamil on the voltage
dependence of the inactivation of WT and the Y652 HERG current
was assessed using the 3-step
protocol[13] shown in the
inset of Figure 4C. The steady-state inactivation curve was
fitted to a Boltzmann equation (Figure 4C, 4D). Peak current
amplitudes were prominently reduced by verapamil (10
µmol/L). For WT HERG channels, the half-point inactivation value
was -27.17±4.58 mV (control) and -48.31±6.10 mV with
verapamil (P<0.001, 6 oocytes), with no significant change
in slope factors (55.9±10.24 and 51.68±8.38 mV for the
control and verapamil, respectively, P>0.05,
n=6). For the Y652A HERG channels, compared with the negative shift of steady
state activation curves by -2 mV, the steady-state
inactivation curves also were shifted to more negative values by -14
mV. The half-point inactivation value was -30.38±3.69 mV
(control) and -44.19±5.68 mV with verapamil
(P<0.001, 6 oocytes), again with no significant change in slope factors
(57.66±8.74 and 59.0232±10.187 mV for the control and
verapamil, respectively, P>0.05;
n=6).
Concentration-dependence blockade of WT and mutant
HERG channels by verapamil Previous reports have shown
that the mutation of Y652 and
F656 to A in the pore helix reduced the blockade of HERG by MK-499, a
methane-sulfonanilide antiarrhythmic
drug[8]. Mutation of the S6 residues also greatly reduced channel blockade by other
compounds such as terfenadine, cisapride, and
chloroquine[8,14]. Therefore, we determined the concentration-effect
relationship for verapamil on Y652A and F656A HERG channels and
compared the potency for blockade with that of the WT HERG
channel. Representative traces were recorded in an
experiment in which a range of concentrations of verapamil were
applied sequentially in the same cell. Current amplitudes
were monitored during the control periods and 10 min of
each drug application with the same voltage protocol. A
total of 5 different concentrations of verapamil were tested.
The protocol used to study the effects of verapamil on WT
and Y652A IHERG is shown in Figure 5B (lower trace). From a
holding potential of -90 mV, the membrane potential was
stepped to 0 mV for 2 s, and then repolarized to -60 mV for 2 s
before returning to -90 mV. The effect of verapamil on the
WT and Y652A HERG channel current is shown in Figure
5A, 5B. Data from individual cells were pooled to obtain the
mean (±SEM) fractional block values, which were then
plotted against the corresponding verapamil concentration as
shown in Figure 5C. Values calculated for tail currents were
then fitted with a Hill equation. The half-maximal
IC50 was 5.1±1.2 µmol/L for WT and 79.6±16 µmol/L for Y652A.
To increase the amplitude of poorly expressed F656A
mutant channels for both WT and F656A, 30 mmol/L
extracellular K+ were used, and tail currents were recorded at -90
mV (Figure 5D, 5E). Using this protocol, the
IC50 was 114±
29 µmol/L for the WT current and 2000±120 µmol/L for the
F656A HERG current (Figure 5F). Thus, mutation of F656A
caused a 20-fold reduction in drug potency, whereas
mutation of Y652A reduced potency by a factor of approximately
16. Verapamil blockade was relieved slowly and incompletely
upon removal of verapamil. Control values were not reached
even after wash-out times of more than 10 min
(n=6 oocytes).
Discussion
The cardinal feature of this study is that verapamil
preferentially binds to and blocks open HERG channels, and we
indicate for the first time that 2 aromatic residues (Tyr-652 or
Phe-656) located in the S6 domain of HERG are critical in the
verapamil-binding site.
Blockade of HERG channels by verapamil In defining
the biophysical properties of the verapamil blockade of WT
and the Y652A HERG channel expressed in
Xenopus oocytes, we found that the midpoint potential
(V0.5) of the activation curves and the inactivation curves were shifted in
the hyperpolarizing direction, indicating that verapamil
altered both channels; the voltage-dependence of both
activation and inactivation gating of the HERG channel shifted
to more negative values. In addition, the degree of channel
blockade directly correlates with channel activation and
verapamil seems to be an open channel blocker of the HERG
channel[9]. In our study, the fractional block increased
rapidly following membrane depolarization, but reached a
plateau after 1 s, indicating time-dependence of inhibition upon
depolarization (Figure 3). This is also evident in Figure 2
where the blockade increased significantly over the voltage
range (negative to -20 mV) and the HERG channel activated
and became saturated at voltages positive to +20 mV (eliciting
maximal HERG channel activation). These results suggest
that channel activation is required for verapamil blockade,
and channel inactivation has little effect on drug affinity.
Previous reports have shown that the voltage
dependence for the blockade of HERG channels by chloroquine
was reversed by the Y652A mutation[14]. In their study, the
blockade of the WT HERG current by chloroquine was
enhanced by progressive depolarization. In contrast, the
blockade of the Y652A HERG current by this drug was diminished
by increased depolarization. These findings suggest that
the interaction of chloroquine with the phenol of Y652
mediates a voltage-dependent blockade of WT HERG channels.
However, in our study, we found that Y652A did not alter the
voltage-dependent block of HERG channels. The blockade
of the WT HERG current by verapamil was enhanced by
progressive depolarization. Similarly, the blockade of the
Y652A HERG current by verapamil was enhanced by
increas-ed depolarization (Figure 2I, 2J).
To examine whether key molecular determinants of HERG
blockade for previously investigated drugs are also
important for HERG channel block by verapamil, we compared the
potency of channel block for WT and 2 mutant HERG
channels (Y652A and F656A) expressed in oocytes. Verapamil
blocked the WT HERG current in a
concentration-dependent manner with an
IC50 value of 5.1 µmol/L at 0 mV (Figure
5). The potency of channel blockade by verapamil was
dramatically reduced in 2 HERG mutant channels
(IC50 was
increased by 16-and 20-fold at 0 mV for Y652A and F656A,
respectively). Our results are consistent with previously
reported studies in which all tested HERG-blocking drugs
(except fluvoxamine) have been shown to block the mutant
HERG channel (F656A) with potency being reduced by over
100-fold, compared to the blockade of the WT HERG channel.
Similarly, mutation of Tyr-652 to alanine also leads to
dramatic attenuation of HERG blockade by most studied drugs
except vesnarinone and
fluvoxamine[8,14_17]. As already stated,
2 amino acids located on the S6 transmembrane domain
(Y652 and F656) are demonstrated to be important in high-affinity
blockade for a number of
compounds[8,18]. Other voltage-gated
K+ channels (Kv1_Kv4) have Ile and Val (Ile) in the
equivalent positions of the aromatic residues Y652 and F656
of HERG. In 2000, Mitcheson and colleagues showed that
MK-499 interacted with the HERG channel, and that
electrostatic interactions between p electrons and hydrogen atoms
of the aromatic rings of Y652/F656 and the drug molecule
were crucial for high-affinity
binding[12]. Similarly, in our study, for alanine-scanning mutagenesis of
Y652 and F656, the potency of channel block by verapamil was dramatically
reduced. These results indicate that Tyr-652 and Phe-656
are critical for the verapamil-induced blockade of the HERG
channel. These findings suggest a possible structural
explanation as to how so many commonly used medications
block HERG, but not other Kv channels and should facilitate
the rational design of drugs devoid of HERG
channel-binding activity.
A previous study reported that the sensitivities of both
WT HERG and an inactivation deficient mutant to the
methanesulphonanilide E-4031 were lowered by a similar
amount of high
(K+)e[19]. The authors concluded this effect
was likely to be due to a direct interaction: electrostatic
repulsion or a "knock-off"
process[19]. It seems reasonable to propose that a similar mechanism accounts for the
observations regarding the effect of 30 mmol/L
(K+)e in Figure 5D of the present study.
Clinical significance It is widely believed that most
drugs associated with Torsade de pointes in humans are
also associated with the HERG K+ channel blockade at
concentrations close to or superimposed upon the free plasma
concentrations found in clinical
use[20]. Verapamil, an L-type calcium antagonist, blocks native and cloned L-type
Ca2+ channels with IC50 values ranging from 250 nmol/L to at least
15.5 µmol/L[21]. It is also reported that verapamil blocks the
WT HERG channels in a concentration range similar to that
required for the blockade of L-type
Ca2+ channels[10]. WT HERG is expressed in HEK 293 cells with an estimated
IC50 of 143.0 nmol/L[10], and 3.8±0.2 µmol/L expressed in
oocytes[11]. Here in our study, verapamil blocked the WT HERG current
in a concentration-dependent manner with an
IC50 value of 5.1 µmol/L at 0 mV (Figure 5). The reduced potency of the drug
blockade in oocytes compared with mammalian cells is a
common finding and may be related to the lipophilic yolk sac in
oocytes that acts to sequester the
drug[4,16]. However, vera-pamil does not cause QT prolongation in clinical studies.
This could be explained by its multiple interactions with
cardiac ion channels. That is, verapamil, which does not
cause long-QT syndrome, may counteract the potential of
the HERG channel blockade which induces QT prolongation
early after depolarization generation through its blockade of
L-type Ca2+ channels.
To the best of our knowledge, verapamil has been
investigated for the first time in our studies that have been
designed to examine the importance of the molecular
interactions between the key S6 residues and drug molecules. A
large number of drugs have been shown to block the HERG
channel current[4,5]. So far, however, the HERG
channel-binding site has only been investigated for a small number of
drugs[8,14_17,22]. More data (well-characterized HERG blockers)
are needed to implement the database, which can be used to
generate a pharmacophore model[23]. The pharmacophore
model may be useful in the pre-synthetic virtual screening of
discovery compounds for HERG activity. Although our data
provides some insights into the mechanisms of interactions
between various drugs and HERG channels, there are still
some limitations in our study. To answer the question as to
why drugs with diverse structure could inhibit
IHERG, extensive site-directed mutagenesis is required in further studies.
Acknowledgements
We are grateful to Dr Michael SANGUINETTI (Utah
University) for the HERG clone.
References
1 Warmke JW, Ganetzky B. A family of potassium channel genes
related to eag in Drosophila and mammals. Proc Natl Acad Sci
USA 1994; 91: 3438_42.
2 Sanguinetti MC, Jiang C, Curran ME, Keating MT. A
mechanistic link between an inherited and an acquired cardiac arrhythmia:
HERG encodes the IKr potassium channel. Cell 1995; 81:
299_307.
3 Roden DM. Current status of class III antiarrhythmic drug
therapy. Am J Cardiol 1993; 72: 44B_9.
4 Cavero I, Mestre M, Guillon JM, Crumb W. Drugs that prolong
QT interval as an unwanted effect: assessing their likelihood of
inducing hazardous cardiac dysrhythmias. Exp Opin
Pharma-cother 2000; 1: 947_73.
5 De Ponti F, Poluzzi E, Cavalli A, Recanatini M, Montanaro N.
Safety of non-antiarrhythmic drugs that prolong the QT
interval or induce torsade de pointes. Drug Safety 2002; 25: 263_86.
6 January CT, Gong Q, Zhou Z. Long QT syndrome: cellular basis
and arrhythmia mechanism in LQT2. J Cardiovasc Electrophysiol
2000; 11: 1413_8.
7 Tseng GN. I (Kr): the hERG channel. J Mol Cell Cardiol 2001;
33: 835_49.
8 Mitcheson JS, Perry MD. Molecular determinants of
high-affinity drug binding to hERG channels. Curr Opin Drug Discov Dev
2003; 6: 667_74.
9 Chouabe C, Drici MD, Romey G, Barhanin J, Lazdunski M. HERG
and KvLQT1/IsK, the cardiac K+ channels involved in long QT
syndromes, are targets for calcium channel blockers. Mol Pharm
1998; 54: 695_703.
10 Zhang S, Zhou ZF, Gong QM, Makielski JC, Craig T. Mechanism
of block and identification of the verapamil binding domain to
HERG potassium channels. Circ Res 1999; 84: 989_98.
11 Waldegger G, Niemeyer K, Mörike CA, Wagner H, Suessbrich AE,
Busch F, et al. Effect of verapamil enantiomers and metabolites
on cardiac K+ channels expressed in
Xenopus oocytes. Cell Physiol Biochem 1999; 9: 81_9
12 Mitcheson JS, Chen J, Lin M, Culberson C, Sanguinetti MC. A
structural basis for drug-induced long QT syndrome. Proc Natl
Acad Sci USA 2000; 97: 12329_33.
13 Su Z, Martin RL, Cox BF, Gintant GA. Ziprasidone: an open
channel blocker of human ether-a-go-go-related gene
K+ channel. J Mol Cell Cardiol 2004; 36: 151_60.
14 Sanchez-Chapula JA, Navarro-Polanco RA, Culberson C, Chen J,
Sanguinetti MC. Molecular determinants of voltage dependent
human ether-a-go-go related gene (HERG) K+
channel block. J Biol Chem 2002; 277: 23587_95.
15 Lees-Miller JP, Duan Y, Teng GQ, Duff HJ. Molecular
determinant of high-affinity dofetilide binding to HERG expressed in
Xenopus oocytes: involvement of S6 sites. Mol Pharmacol 2000;
57: 367_74.
16 Amiya K, Mitcheson JS, Yasui K, Kodama I, Sanguinetti MC.
Open channel block of HERG K+ channels by vesnarinone. Mol
Pharmacol 2001; 60: 244_53.
17 Sanchez-Chapula JA, Ferrer T, Navarro-Polanco RA, Sanguinetti
MC. Voltage-dependent profile of human ethera-go-go-related
gene channel block is influenced by a single residue in the S6
transmembrane domain. Mol Pharmacol 2003; 63: 1051_8.
18 Wang S, Morales MJ, Liu S, Strauss HC, Rasmusson RL.
Modulation of HERG affinity for E-4031 by
[K+]o and C-type inactivation,
FEBS Lett 1997; 417: 43_7.
19 Sanguinetti MC, Mitcheson JS. Predicting drug-HERG channel
interactions that cause acquired long QT syndrome. Trends
Pharmacol Sci 2005; 26: 119_24.
20 Redfern WS, Carlsson L, Davis AS, Lynch WG, MacKenzie I,
Palethorpe S, et al. Relationships between preclinical cardiac
electrophysiology, clinical QT interval prolongation and
torsade de pointes for a broad range of drugs: evidence for a provisional
safety margin in drug development. Cardiovasc Res 2003; 58:
32_45.
21 Hosey MM, Lazdunski M. Calcium channels: molecular
pharma-cology, structure and regulation. J Membr Biol 1988; 104:
81_105.
22 Scholz EP, Zitron E, Kiesecker C, Lueck S, Kathofer S, Thomas
D, et al. Drug binding to aromatic residues in the HERG channel
pore cavity as possible explanation for acquired long QT
syndrome by antiparkinsonian drug budipine. Naunyn Schmiedebergs
Arch Pharmacol 2003; 368: 404_14.
23 Cavalli A, Poluzzi E, De Ponti F, Recanatini M. Toward a
pharmacophore for drugs inducing the long QT syndrome:
insights from a CoMFA study of HERG K+ channel blockers. J Med
Chem 2002; 45: 3844_53.
|
