Download The human cardiac K2P3.1 (TASK-1) potassium leak channel is a

Naunyn-Schmied Arch Pharmacol (2010) 381:261–270
DOI 10.1007/s00210-009-0454-4
ORIGINAL ARTICLE
The human cardiac K2P3.1 (TASK-1) potassium leak channel
is a molecular target for the class III antiarrhythmic
drug amiodarone
Jakob Gierten & Eckhard Ficker & Ramona Bloehs & Patrick A. Schweizer &
Edgar Zitron & Eberhard Scholz & Christoph Karle & Hugo A. Katus & Dierk Thomas
Received: 20 April 2009 / Accepted: 4 September 2009 / Published online: 24 September 2009
# Springer-Verlag 2009
Abstract Two-pore-domain (K2P) potassium channels
mediate background potassium currents, stabilizing resting
membrane potential and expediting action potential repolarization. In the heart, K2P3.1 (TASK-1) channels are
implicated in the cardiac plateau current, IKP. Class III
antiarrhythmic drugs target cardiac K+ currents, resulting
in action potential prolongation and suppression of atrial
and ventricular arrhythmias. The objective of this study
was to investigate acute effects of the class III antiarrhythmic drug amiodarone on human K2P3.1 channels.
Potassium currents were recorded from Xenopus oocytes
using the two-microelectrode voltage clamp technique.
Amiodarone produced concentration-dependent inhibition
of hK2P3.1 currents (IC50 = 0.40 µM) with maximum
current reduction of 58.1%. Open rectification properties
that are characteristic to hK2P3.1 currents were not altered
by amiodarone. Channels were blocked in open and closed
states in reverse frequency-dependent manner. hK2P3.1
channel inhibition was voltage-independent at voltages
between −40 and +60 mV. Modulation of protein kinase C
activity by amiodarone does not contribute to hK2P3.1
current reduction, as pre-treatment with the protein kinase
C inhibitor, staurosporine, did not affect amiodarone
J. Gierten : R. Bloehs : P. A. Schweizer : E. Zitron : E. Scholz :
C. Karle : H. A. Katus : D. Thomas (*)
Department of Cardiology,
Medical University Hospital Heidelberg,
Im Neuenheimer Feld 410,
69120 Heidelberg, Germany
e-mail: [email protected]
E. Ficker
Rammelkamp Center, MetroHealth Campus,
Case Western Reserve University,
2500 MetroHealth Drive,
Cleveland, OH 44109, USA
block. Amiodarone is an inhibitor of cardiac hK2P3.1
background channels. Amiodarone blockade of hK2P3.1
may cause prolongation of cardiac repolarization and
action potential duration in patients with high individual
plasma concentrations, possibly contributing to the antiarrhythmic efficacy of the class III drug.
Keywords Amiodarone . Antiarrhythmic drug .
Background potassium current . Cardiac arrhythmia .
K2P channel
Introduction
Two-pore-domain potassium (K2P) channels stabilize resting
membrane potential (RMP) below firing threshold and
expedite repolarization of action potentials (Goldstein et al.
2001). Because membrane potential is fundamental to
cardiac activity, leak current regulation is a primary and
dynamic mechanism for control of cellular excitability
(Goldstein et al. 2001; Patel and Honore 2001; Bayliss et
al. 2003; Thomas et al. 2008). K2P channels are identified by
a unique structure of two pore-forming loop domains in each
subunit. The channels assemble from two subunits to form
an ion conduction pathway. In the heart, repolarization of the
action potential is mediated by multiple potassium conductances (Nerbonne and Kass 2005). The cardiac plateau
current, IKP, is a rapidly activating, non-inactivating potassium current that regulates amplitude and duration of the
cardiac action potential (Backx and Marban 1993; Marban
2002). On the basis of common distribution and biophysical
attributes, it has been suggested that K2P3.1 (TASK-1)
channels contribute to IKP (Lopes et al. 2000; Nerbonne
and Kass 2005). K2P3.1 is expressed in mouse and human
heart (Duprat et al. 1997; Lopes et al. 2000). Endogenous
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K2P3.1-like currents have been detected in rat cardiomyocytes, and inhibition of these currents causes prolongation of
the cardiac action potential (Putzke et al. 2007).
Prolongation of cardiac refractory period and action
potential is the hallmark of class III antiarrhythmic
drugs, resulting in reduced membrane excitability and
decreased arrhythmia susceptibility. Amiodarone is one
of the most effective class III antiarrhythmic agents for
the management of ventricular and supraventricular
tachyarrhythmias (Naccarelli et al. 2000; Zimetbaum
2007). Class III substances like amiodarone are potent
potassium channel blockers that act primarily through
inhibition of the rapid component of the cardiac delayed
rectifier potassium current, IKr, and the underlying human
ether-à-go-go related gene (hERG) potassium channel
(Kiehn et al. 1999; Kathöfer et al. 2005). Amiodarone is
associated with a relatively low proarrhythmic potential,
probably due to its multiple pharmacological actions on
different cardiac ion channels and receptors (Podrid 1995).
Effects of amiodarone on the cardiac plateau current, IKP,
or underlying K2P potassium channels have not been
investigated to date. This study was designed to assess
acute effects of amiodarone on human K2P3.1 channels in
order to further elucidate the electrophysiological profile
of the drug. Here, we describe amiodarone inhibition of
hK2P3.1 background potassium channels. hK2P3.1 current
blockade by amiodarone is expected to cause action
potential prolongation in patients with high individual
plasma drug concentrations, thereby contributing to the
class III antiarrhythmic action of amiodarone.
Methods
Molecular biology
Human complementary DNA (cDNA) clone encoding
K2P3.1 (NM_002246) was provided by Dr. Steve Goldstein
(Chicago, IL, USA) in pRAT, a dual-purpose expression
vector containing a cytomegalovirus promoter for mammalian expression and a T7 promoter for cRNA synthesis.
This study has been carried out in accordance with the
Guide for the Care and Use of Laboratory Animals as
adopted and promulgated by the US National Institutes of
Health (publication number 86-23, revised 1985), and the
current version of the German Law on the Protection of
Animals was followed. Procedures for in vitro transcription
and oocyte injection were performed as published previously (Kiehn et al. 1999). Briefly, complementary RNAs
(cRNAs) were transcribed after vector linearization using
T7 RNA polymerase and the mMessage mMachine kit
(Ambion, Austin, TX, USA). Transcripts were quantified
using a spectrophotometer and by comparison with control
Naunyn-Schmied Arch Pharmacol (2010) 381:261–270
samples separated by agarose gel electrophoresis. Stage VVI defolliculated Xenopus oocytes were injected with 46 nl
of cRNA per cell.
Electrophysiology
Two-electrode voltage clamp measurements were performed as described earlier (Thomas et al. 1999). Whole
cell currents were measured 2 to 3 days after injection
with an Oocyte Clamp amplifier (Warner Instruments,
Hamden, CT, USA) using pCLAMP (Axon Instruments,
Foster City, CA, USA) and Origin (OriginLab, Northampton, MA, USA) software for data acquisition and
analysis. Data were sampled at 2 kHz and filtered at
1 kHz. All experiments were carried out at room
temperature (20–22°C), and no leak subtraction was done
during the experiments.
Solutions and drug administration
Two-electrode voltage clamp electrodes were filled with
3 M KCl and had tip resistances of 1 to 5 MΩ.
Recordings were performed under constant perfusion at
room temperature. The standard physiological extracellular solution contained 96 mM NaCl, 4 mM KCl,
1.1 mM CaCl2, 1 mM MgCl2, and 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid. pH was adjusted
to 7.4 with NaOH. Amiodarone (2-butyl-3-benzofuranyl4-[2-(diethylamino)ethoxy]-3,5-diiodophenyl-ketone) hydrochloride (Sigma) was dissolved in ethanol to a stock
solution of 10 mM and stored at +4°C. Reduced drug
solubility was observed at 100 μM bath concentration.
Thus, hK2P3.1 current reduction induced by 100 μM
amiodarone may be slightly underestimated in this study.
Application of 1% (v/v) ethanol (the maximum bath
concentration) for 30 min reduced hK2P3.1 currents
measured at the end of the +20 mV-test pulse by 19.9±
3.4% (n= 5; p= 0.02), whereas a time control period
(30 min) did not significantly alter current amplitudes
measured as described in Fig. 1a (Δcurrent=3.5±2.5%;
n=4; p=0.36). To equilibrate hK2P3.1 currents prior to
drug application, the solvent was added to the bath
solution at corresponding concentrations during all control
periods and during staurosporine pre-treatment of oocytes.
Staurosporine (Sigma) was dissolved in dimethyl sulfoxide to a stock solution of 2 mM and stored at −20°C. On
the day of experiments, aliquots of the stock solution were
diluted to the desired concentration with the bath solution.
Data analysis and statistics
Concentration-response relationships for drug-induced block
were fitted with a Hill equation of the following form: Idrug/
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Icontrol =1/[1+(D/IC50)n], where I indicates current, D is the
drug concentration, n is the Hill coefficient, and IC50 is the
concentration necessary for half-maximal block. Data are
expressed as mean±SEM. We used Student’s t tests (twotailed tests) to compare statistical significance of the results:
p<0.05 was considered statistically significant. When more
than two samples were compared (Fig. 4e), statistical
significance was first assessed by one-way analysis of
variance (ANOVA). If the hypothesis of equal means among
multiple samples could be rejected at the 0.05-level according to ANOVA, the statistically significant effect was further
analyzed in order to assess which samples are different from
each other using a second follow-up, post hoc test: pair wise
comparisons of groups were made using Student’s t test, and
the probability values were adjusted for multiple comparisons using the Bonferroni correction.
Results
Amiodarone inhibits hK2P3.1 (TASK-1) potassium leak
channels
The effects of amiodarone on human K2P3.1 channels were
studied in Xenopus laevis oocytes. Amiodarone reduced
hK2P3.1 potassium currents in a concentration-dependent
manner, as displayed in Fig. 1. Currents were elicited by a
500-ms depolarizing step to +20 mV and measured at the
end of the test pulse, and the degree of block was
determined after 30 min (Fig. 1a). The holding potential
was −80 mV in all experiments performed in this study. To
study concentration-dependence of hK2P3.1 inhibition by
amiodarone, currents in the presence of the drug were
normalized to their respective control values and plotted as
relative current amplitudes in Fig. 1b (n=4 to 13 cells were
investigated at each concentration). Calculation of the halfmaximal inhibitory concentration (IC50) for block of
hK2P3.1 leak channels yielded 0.40±0.04 μM with a Hill
coefficient nH of 1.12±0.10. However, blockade was not
complete, even with 100 μM amiodarone. The onset of
block is shown in Fig. 1c (n=7). After a control period of
30 min showing current decrease induced by the solvent,
ethanol (see “Methods” section), hK2P3.1 current reduction
by 100 μM amiodarone developed rapidly. Upon washout
(8 min), inhibitory effects of amiodarone on hK2P3.1 were
only partially reversible.
Figure 1a illustrates that hK2P3.1 channels activate in
two phases (Duprat et al. 1997). The term “two phase
activation” refers to the observation that currents activate
quickly to approximately 85% of their respective maximum
amplitudes within approximately 50 ms, followed by
markedly slower additional activation time course. Macroscopic hK2P3.1 currents can be divided into an instanta-
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neous (measured 1.5 ms after the step to +20 mV) and a
time-dependent current (measured at the end of the 500 mstest pulse), respectively. The instantaneous current was
77.3±2.4% of the fully activated current under control
conditions (n=7). The difference between inhibition of the
instantaneous component (44.1±3.3% current reduction)
and the total current (50.4±3.2% inhibition) by 100 μM
amiodarone (30 min) was not significantly different
(Fig. 1d).
hK2P3.1 currents recorded in physiological saline solution revealed electrophysiological characteristics typical for
a potassium-selective background leak conductance, that is,
a voltage-independent portal showing Goldman–Hodgkin–
Katz, or open, rectification (Fig. 2a; Goldstein et al. 2001).
Potassium channels that display open rectification pass
current more readily in one direction (rectify) owing to
unequal ion concentration across the membrane. To study
the effects of amiodarone on hK2P3.1 rectification, linear
ramp voltage protocols were applied between −140 and
+60 mV (500 ms) before and after application of 100 μM
amiodarone for 30 min (Fig. 2a). Both currents showed
similar outward rectification. The degree of block determined at +20 mV ramp potential was 46.2±2.7% (n=4). In
this series of experiments, a control period of 30 min
(Fig. 2b) revealed current decrease by 8.1±1.8% (n=6; p=
0.01). Application of the solvent (1% ethanol; Fig. 2c)
induced similar current reduction (8.2±1.3%; n=5; p=
0.01), consistent with weak time-dependent rundown of
hK2P3.1 ramp currents.
Amiodarone-induced reduction of hK2P3.1 leak current
magnitude was accompanied by changes in RMP (Fig. 2d).
Compared to control recordings (RMP=−61.7±1.3 mV),
amiodarone (100 μM, 30 min) depolarized Xenopus oocyte
RMP by 9.9±1.6 mV to −51.8±2.4 mV (n=11; p=0.002).
Figure 2e and f illustrate that a 30-min-control period
(ΔRMP= 0.6 ± 0.3 mV; n = 5; p = 0.75) or the solvent,
ethanol, at its maximum bath concentration (1%; ΔRMP=
0.7±0.1 mV; n=5; p=0.73) did not cause membrane
potential depolarization.
Amiodarone blocks open and closed hK2P3.1 channels
Despite being a leak channel that is open over the entire
physiological voltage range, hK2P3.1 currents show some
voltage-dependence with increased activation rates (i.e.,
faster transitions from closed to open states of single K2P3.1
channels) at more positive potentials (Lopes et al. 2000). To
investigate whether there is a difference of channel
sensitivity in the closed or open state, we recorded
hK2P3.1 currents during a single depolarizing step to
+20 mV for 7.5 s. Typical current traces under control
conditions and after application of 100 μM amiodarone for
30 min while holding the cell at −80 mV are displayed
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Fig. 1 Inhibition of human
K2P3.1 (TASK-1) channels by
amiodarone. Representative
current traces recorded from the
same cell under control
conditions and after application
of amiodarone (100 μM,
30 min) are displayed in panel
a. b Concentration-response
relationships for the effect of
amiodarone on hK2P3.1 outward
currents measured at the end
of the +20 mV voltage step
(n=4 to 13 cells). The IC50
yielded 0.40 μM, and maximum
current inhibition was 58.1%.
c Time course of hK2P3.1
current inhibition by 100 μM
amiodarone (n=7).
d Amiodarone blockade of the
instantaneous and sustained
components of hK2P3.1 current
is not statistically different
(n=7). Data are given as
mean±SEM. Dotted line
indicates zero current level
(Fig. 3a). The degree of inhibition (i.e., (1-current in the
presence of amiodarone/control current)×100) after the
incubation period is displayed with linear and logarithmic
time scales in Fig. 3b and c, respectively. During the
+20 mV-step, the fraction of channels in the open state is
expected to be larger compared to −80 mV. Analysis of the
test pulse after amiodarone administration revealed that
pronounced inhibition of hK2P3.1 channels had already
occurred at −80 mV (39.2±1.7%; n=4; p=0.0008), and
weak additional time-dependent inhibition was observed
during the +20 mV-pulse (block at the end of the test pulse:
48.1±1.7%; n=4; p=0.0004). Corresponding time control
experiments (Fig. 3d) revealed weak current rundown by
7.0 ± 1.6% (n = 6; p = 0.008). Figure 3e illustrates that
ethanol application (1%) did not significantly affect
hK2P3.1 currents measured at the end of the 7.5-s test pulse
(Δcurrent=2.4±1.1%; n=5; p=0.10).
Voltage-dependence of hK2P3.1 current blockade
by amiodarone
The effect of amiodarone on hK2P3.1 current voltage (I-V)
relationship was investigated under isochronal recording
conditions. From a holding potential of −80 mV, depolarizing pulses were applied for 500 ms to voltages between
−140 and +60 mV in 20 mV increments (0.5 Hz). Families
of current traces from one cell are shown for control
conditions (Fig. 4a) and after exposure to 100 μM
amiodarone for 30 min (Fig. 4b). There was no apparent
shift in the current–voltage relationship after amiodarone
administration (Fig. 4c, d). Relative inhibition of hK2P3.1
currents was plotted as function of the test pulse potential in
Fig. 4e (n=8). Amiodarone reduced hK2P3.1 currents
between −40 and +60 mV without marked differences in
the degree of blockade. In contrast, the effect of amiodarone was significantly reduced at potentials below the
reversal potential with voltage-dependent increase of block
from −140 to −100 mV. It has to be taken into consideration
that inward currents of outwardly rectifying hK2P3.1
channels are relatively low under the given experimental
conditions. Thus, data on relative block of inward currents
have to be interpreted with care.
Reverse frequency-dependence of amiodarone-induced
hK2P3.1 block
To study frequency-dependence of block, hK2P3.1 channels
were rapidly activated by a depolarizing step to +20 mV
(500 ms) at intervals of 1 or 10 s, respectively, with each
cell studied at only one stimulation rate. Five oocytes were
used at each rate, and the development of current reduction
in the presence of 100 μM amiodarone was plotted versus
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265
Fig. 2 Rectification of hK2P3.1 current elicited by voltage ramps
from −140 to +60 mV. a Typical recordings from the same cell in the
absence of the drug and after superfusion with 100 μM amiodarone
(30 min) are superimposed. Dotted lines indicate zero current level.
Time (30 min) and solvent (1% ethanol) controls are shown in panels
b and c, respectively. d Mean resting membrane potentials (RMP) of
Xenopus oocytes, measured before and after blockade of hK2P3.1 with
amiodarone (100 μM, 30 min). Leak current inhibition depolarized the
cell membrane by 9.9 mV (n=11). e, f Time (n=5) and solvent (n=5)
controls, corresponding to data presented in panel d. Data are given as
mean±SEM. Double asterisks, p<0.01
Fig. 3 Blockade of open and closed hK2P3.1 channels. Currents were
activated by a 7.5-s depolarizing voltage step to +20 mV. a
Representative control recording and the first pulse measured
immediately after administration of 100 μM amiodarone (30 min)
are shown. b and c display the degree of current inhibition in percent
(b, linear time scale; c, logarithmic time scale), demonstrating
blockade of closed and open hK2P3.1 channels. Similar results were
obtained from four independent experiments. Representative time and
solvent control experiments from series of n=6 and n=5 cells are
depicted in panels d and e, respectively
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Fig. 4 Effects of amiodarone on hK2P3.1 voltage-dependence of
activation. Control measurement (a) and the effect of 100 μM
amiodarone (30 min; b) are shown in one representative oocyte. Zero
current levels are indicated by dotted lines. Panels c and d display
activation curves, i.e., step current amplitudes as function of test
potentials, recorded under isochronal conditions (c, original current
amplitudes; d, values normalized to maximum currents) (n=8). e The
fraction of blocked step currents is plotted as function of the respective
test pulse potential. Channel block displayed significant differences
between potentials below the reversal potential (−140 to −100 mV) and
positive of the reversal potential (−40 to +60 mV), respectively (n=
8 cells). Data are expressed as mean±SEM. Single asterisk, p<0.05,
double asterisk, p<0.01 versus relative block at +60 mV
time (Fig. 5a). The degree of inhibition after 30 min was
significantly (p=0.0007) higher at 1 Hz stimulation rate
(81.9±1.9%; n=5; p=0.008) compared to 0.1 Hz (62.4±
3.2%; n=5; p=0.0005). In contrast, no statistically significant current reduction was measured during a time control
period of 30 min (1 Hz: −10.2±7.9%; n=5; p=0.83;
0.1 Hz: −7.6±1.9%; n=5; p=0.72; Fig. 5b). However,
frequency-dependent current inhibition was observed upon
incubation with the solvent ethanol (1%; 30 min) as well
(Fig. 5c). At 1 Hz stimulation rate, currents were blocked
by 63.7±10.1% (n=5; p=0.005). Ethanol-associated current inhibition at 0.1 Hz was significantly (p=0.002) lower
(18.4±2.2%; n=5; p=0.02). Subsequent correction for
ethanol block yielded more pronounced amiodarone-
induced current inhibition at lower stimulation rates
(44.0% block; 0.1 Hz) compared to higher rates (18.2%
block; 1 Hz), revealing reverse frequency-dependence of
hK2P3.1 inhibition by amiodarone.
Inhibition of hK2P3.1 currents is independent of protein
kinase C activity
In addition to direct blockade, amidarone may affect ion
channel function indirectly by regulating protein kinase C
(PKC) activity (Silver et al. 1989; Futamura 1996). To
determine whether PKC contributes to amiodarone inhibition of hK2P3.1, cells were incubated with the PKC
inhibitor, staurosporine (1 µM), for 0.5 to 3 h prior to
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267
Fig. 5 Amiodarone block of hK2P3.1 depends on stimulation rate.
Panels a–c show the effects of 100 µM amiodarone (a), no specific
treatment (b), and 1% ethanol (c), respectively. Mean relative hK2P3.1
current amplitudes recorded at +20 mV membrane potential (1 and
0.1 Hz stimulation rate) are plotted versus time (n=5 oocytes were
studied at each rate; error bars denote SEM; double asterisk, p<0.01).
For the purpose of clear presentation, not all measurements are
displayed
electrophysiological recordings (Fig. 6). Currents were
recorded using the protocol described in Fig. 4 and
measured at the end of the test pulse to +20 mV. Following
staurosporine pre-treatment, oocytes were superfused with
100 μM amiodarone for 30 min. There were no significant
changes in the degree of block after PKC inhibition by
staurosporine (56.3 ± 3.8%; n = 6) when compared to
oocytes from the same batch without staurosporine pretreatment (58.7±5.0%; n=4), arguing against a significant
role of PKC in amiodarone inhibition of hK2P3.1.
inhibition may not be clinically relevant in the majority of
patients treated with amiodarone. However, in patients with
relatively high individual free plasma concentrations,
blockade of hK2P3.1 leak currents by amiodarone may
contribute to electrophysiological action of the drug.
Pharmacological inhibition of K2P3.1 channels has been
reported previously for the class I antiarrhythmic, quinidine, and several non-cardiac drugs, including local and
volatile anesthetics and psychotropic drugs (Table 1). To
our knowledge, the present study is the first report of K2P
current blockade by a class III antiarrhythmic drug.
Discussion
The biophysical mechanism of hK2P3.1 inhibition
Acute effects of amiodarone on human K2P3.1 leak
channels
The rapid onset of block argues in favor of a direct drugchannel interaction and against increased protein turnover
or accelerated protein degradation as molecular mechanisms of action. Unblocking occurred rather slowly, and a
marked washout could not be achieved. The apparent
irreversibility of block may be attributed in the first instance
to trapping of the drug molecule inside the hK2P3.1 channel
pore cavity. It has to be taken into consideration that this
hypothesis remains speculative until the three-dimensional
structure of K2P channels is resolved. Intracellular accumulation of the lipophilic drug molecule may further contribute to extremely slow washout kinetics. The primary
mechanism underlying this phenomenon is lysosomal
amiodarone accumulation via pH trapping. The amine
amiodarone enters lysosomes in unprotonated form, where
protonation prevents drug efflux. In addition, amiodarone
may accumulate in mitochondria, further increasing intracellular amiodarone content. The long elimination half life
of the drug additionally supports amiodarone tissue
accumulation.
Human K2P3.1 two-pore-domain potassium channels are
blocked by the class III antiarrhythmic drug amiodarone.
Inhibition of hK2P3.1 channels expressed in Xenopus
oocytes displayed an IC50 value of 0.40 μM with maximum
current reduction of 58.1%. Mean therapeutic amiodarone
plasma concentrations have been estimated as between 1
and 3 μM, with concentrations ranging from 0.15 to
18.4 µM (Haffajee et al. 1983; Latini et al. 1984).
Amiodarone is highly lipophilic, leading to drug accumulation in cardiac tissue. In contrast, extensive plasma
protein binding (approximately 99.8%) reduces free, biologically active amiodarone (Veronese et al. 1988). Thus,
free amiodarone levels are expected to yield 0.3–370 nM,
revealing a 1.1 to 1,333-fold difference compared to the
hK2P3.1 IC50 value obtained here. Taking into consideration that mean concentrations (1–3 µM) differ from
experimental IC50 value by 67–200-fold, leak channel
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Fig. 6 Protein kinase C (PKC) is not involved in amiodarone
inhibition of hK2P3.1. Oocytes were treated with 1 µM staurosporine,
a PKC inhibitor, for 0.5–3 h prior to current recordings. The effect of
100 µM amiodarone (30 min) was not significantly altered by pretreatment with staurosporine (n=6) in comparison to control cells
(Mock; n=4). Data are given as mean±SEM
Reverse frequency-dependence with reduced block at
higher stimulation rates was observed (Fig. 5). This finding
could be explained by different degrees of inhibition and/or
unblocking that occur during the time in between pulses (i.e.,
at −80 mV when no macroscopic current is detected and open
probability is low), possibly indicating higher drug affinity to
closed channels. In addition, the inhibitory action of amiodarone was voltage-independent at voltages between −40
and +60 mV, that is, current inhibition occurred with similar
potency at these membrane potentials (Fig. 4). Reduced
relative hK2P3.1 inhibition was seen at more negative
membrane potentials, as previously observed with amiodar-
one block of hERG channels (Kiehn et al. 1999). More
efficient blockade at positive potentials could be explained as
follows. Membrane depolarization may cause movement of
the positively charged drug molecule into the central channel
pore cavity, possibly increasing drug binding to a hypothetical depolarization-favored binding site. Future studies
assessing structural determinants of the K2P3.1 drugbinding site are necessary in order to further evaluate this
hypothesis.
hK2P3.1 channels mediate leak conductances open across
the entire physiological voltage range. Open rectification, a
biophysical property characteristic to hK2P3.1 function in
physiological ionic conditions, was not altered by amiodarone (Fig. 2). K2P3.1 channels display gating (i.e., opening
and closing of single channels) and show voltage- and timedependent responses to changes in membrane potential
(Lopes et al. 2000) with greater activation rates and faster
transitions from closed to open states of single channels at
more positive membrane potentials. This mechanism could
explain the presence of an instantaneous and a timedependent current component (Figs. 1a, d and 3a). The fact
that both current components are markedly blocked by
amiodarone suggests that the drug may bind to hK2P3.1 in its
open and closed channel states (Fig. 3). However, we
observed a small difference in block of instantaneous and
time-dependent current components. In Fig. 1d, this difference was not statistically significant. On a larger time scale
(7.5 s voltage pulse in Fig. 3a), blockade of time-dependent
hK2P3.1 current was 8.9% greater than inhibition of
instantaneous currents. It is noteworthy that we cannot
Table 1 Pharmacology of K2P3.1 (TASK-1) channels
Drug
Effect
IC50/EC50
Maximum effect
A293
Amiodarone
Bupivacaine
Etidocaine
Genistein
Inhibition
Inhibition
Inhibition
Inhibition
Inhibitiona
Halothane
Lidocaine
Mepivacaine
Methanandamide
Phenytoin
Activation
Inhibition
Inhibition
Inhibition
Inhibition
0.2 μM (XO)
0.4 μM (XO)
41 μM (XO)
39 μM (XO)
10.7 μM (XO)
12.3 μM (MC)
0.3–0.4 mM (MC)
222 μM (XO)
709 μM (XO)
0.7 μM (MC)
NI
Approximately 95%
Approximately 58%
Approximately 95%
Approximately 90%
Approximately 90%
Approximately 85%
Approximately 60%
Approximately 70%
Approximately 52%
Approximately 99%
53% reduction (200
reduction
reduction
reduction
reduction
reduction
reduction
increase
reduction
reduction
reduction
μM; XO)
Quinidine
R(+)-ropivacaine
S(-)-ropivacaine
Tetracaine
Inhibition
Inhibition
Inhibition
Inhibition
NI
51 μM (XO)
53 μM (XO)
668 μM (XO)
71% reduction (100
Approximately 95%
Approximately 92%
Approximately 52%
μM; XO)
reduction
reduction
reduction
a
Inhibitory effects are at least partially mediated via protein tyrosine kinases
XO Xenopus oocytes, MC mammalian cells, NI not investigated
Reference
Putzke et al. 2007
This study
Kindler et al. 1999
Kindler et al. 1999
Gierten et al. 2008
Sirois et al. 2000
Kindler et al. 1999
Kindler et al. 1999
Maingret et al. 2001
Leonoudakis et al. 1998
Leonoudakis et al. 1998
Kindler et al. 1999
Kindler et al. 1999
Naunyn-Schmied Arch Pharmacol (2010) 381:261–270
distinguish precisely between open and closed states because
the majority of channels are open over the entire voltage
range. Furthermore, unblocking is slow, and drug molecules
that bind to the open state may still occupy the channel in
subsequent open state–closed state transitions. Instantaneous
and time-dependent K2P current components may be
explained by transitions of different closed channel states
(C1 and C2, respectively) to the open conformation, as
suggested previously for K2P2.1 (Honore et al. 2002). If this
hypothesis is true and applies to K2P3.1 channels, it is
reasonable to speculate that hypothetical closed states C1 and
C2 were blocked with similar affinity by amiodarone. We
conclude that amiodarone may bind to closed and open
channel conformations. However, we cannot determine
which state displays higher affinity.
Amiodarone might indirectly affect hK2P3.1 channel
function via modulation of PKC activity and subsequent
alteration of PKC-regulated hK2P3.1 channels (Silver et al.
1989; Futamura 1996; Besana et al. 2004). Here, inhibition
of PKC by preincubation with staurosporine did not alter
amiodarone-induced hK2P3.1 current reduction, suggesting
that PKC-dependent signal transduction pathways are not
involved (Fig. 6). In summary, it is reasonable to assume
that amiodarone inhibits hK2P3.1 channels via direct
binding and blockade of the ion conduction pathway.
Future studies including chimeric approaches and analyses
of the putative drug-binding site in hK2P3.1 are required in
order to characterize the underlying drug-binding mechanism in detail.
269
logical inhibition of K2P potassium currents leads to
membrane potential depolarization. The present study
revealed that amiodarone depolarized the RMP by
9.9 mV, an electrophysiological alteration expected to
promote excitability and to result in cardiac arrhythmogenesis. This hypothesis is in line with a report of
proarrhythmic effects associated with K2P3.1 inhibition
(Barbuti et al. 2002). Further support is provided by the
observation that suppression of the Drosophila K2P channel, ORK1, results in increased cardiac electrical automaticity (Lalevee et al. 2006).
Study limitations
Electrophysiological studies in heterologous expression
systems such as Xenopus oocytes provide valuable information on ion channel electrophysiology and biophysics. It
should be noted, however, that the human cardiac phenotype might display differences, and extrapolations to human
physiology should be handled with appropriate care. We
focused our investigation on hK2P3.1 because, based on
experimental data available to date, this channel is thought
to contribute to the cardiac plateau current, IKP. Anti- and
proarrhythmic effects of amiodarone, however, are likely to
result from combined drug effects on multiple channels and
receptors (Podrid 1995). Furthermore, long-term effects of
amiodarone and its metabolite, desethylamiodarone, may
induce further modulation of cardiac hK2P3.1 channels and
cardiac excitability in addition to acute drug action
investigated in this study.
Clinical implications
During cardiac action potentials at depolarized membrane
voltages, outward potassium currents mediated by K2P
channels contribute to repolarization of cardiomyocytes.
Consequently, inhibition of cardiac K2P3.1 channels is
expected to prolong refractoriness and action potential
duration in the heart. Indeed, Putzke et al. (2007) reported
action potential prolongation upon application of the
K2P3.1 antagonist A293 in isolated rat ventricular cardiomyocytes. We propose that hK2P3.1 current blockade by
amiodarone prolongs cardiac refractoriness and contributes
to the class III antiarrhythmic action of amiodarone in
patients with high individual plasma concentrations.
A common side effect of antiarrhythmic drugs is their
proarrhythmic potential. Under amiodarone therapy, 0.7%
of patients have been reported to develop torsade de
pointes tachycardia (Hohnloser et al. 1994). It is possible
that block of hK2P3.1 channels by amiodarone contributes
to the proarrhythmic potential of amiodarone treatment
owing to the following mechanism. During RMP at
negative voltages, K2P leak channels stabilize the membrane potential and suppress excitability. Here, pharmaco-
Acknowledgments We are grateful to Dr. Steve Goldstein for
providing the cDNA clone encoding hK2P3.1. This work was
supported in part by grants from the Deutsche Forschungsgemeinschaft (project KA 1714/1-1 to C.K.), from the German Cardiac
Society (Max Schaldach Research Scholarship to D.T.), from the
University of Heidelberg (FRONTIERS program), from the
ADUMED-Foundation (to D.T.), and from the National Institutes of
Health (HL71789 to E.F.). J.G. is a fellow of the MD/PhD program at
the University of Heidelberg.
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