Activation of human ether-a-go-go-related gene potassium channels by the diphenylurea 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643)

Article abstract of DOI:10.1124/mol.105.015859

The cardiac action potential is generated by a concerted action of different ion channels and transporters. Dysfunction of any of these membrane proteins can give rise to cardiac arrhythmias, which is particularly true for the repolarizing potassium channels. We suggest that an increased repolarization current could be a new antiarrhythmic principle, because it possibly would attenuate afterdepolarizations, ischemic leak currents, and reentry phenomena. Repolarization of the cardiac myocytes is crucially dependent on the late rapid delayed rectifier current (I_Kr) conducted by ether-a-go-go-related gene (ERG) potassium channels. We have developed the diphenylurea compound 1,3-bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and tested whether this small organic molecule could increase the activity of human ERG (HERG) channels expressed heterologously. In Xenopus laevis oocytes, NS1643 increased both steady-state and tail current at all voltages tested. The EC₅₀ value for HERG channel activation was 10.5 μM. These results were reproduced on HERG channels expressed in mammalian human embryonic kidney 293 cells. In guinea pig cardiomyocytes, studied by patch clamp, application of 10 μM NS1643 activated I_Kr and significantly decreased the action potential duration to 65% of the control values. The effect could be reverted by application of the specific HERG channel inhibitor 4′-[[1-[2-(6-methyl-2- pyridyl)ethyl]-4-piperidinyl]carbonyl]-methanesulfonanilide (E-4031) at 100 nM. Application of NS1643 also resulted in a prolonged postrepolarization refractory time. Finally, cardiomyocytes exposed to NS1643 resisted reactivation by small depolarizing currents mimicking early afterdepolarizations. In conclusion, HERG channel activation by small molecules such as NS1643 increases the repolarization reserve and presents an interesting new antiarrhythmic approach. Copyright

Full text of DOI:10.1124/mol.105.015859

0026-895X/06/6901-266–277$20.00
MOLECULAR PHARMACOLOGY
Copyright © 2006 The American Society for Pharmacology and Experimental Therapeutics
Mol Pharmacol 69:266–277, 2006
Vol. 69, No. 1
15859/3071472
Printed in U.S.A.
Activation of Human ether-a-go-go-Related Gene Potassium
Channels by the Diphenylurea 1,3-Bis-(2-hydroxy-5-
trifluoromethyl-phenyl)-urea (NS1643)
Rie Schultz Hansen, Thomas Goldin Diness, Torsten Christ, Joachim Demnitz,
Ursula Ravens, Søren-Peter Olesen, and Morten Grunnet
NeuroSearch A/S, Ballerup, Denmark (R.S.H., T.G.D., J.D., S.-P.O., M.G.); Danish Arrhythmia Research Centre and Department
of Medical Physiology, Panum Institute, University of Copenhagen, Copenhagen, Denmark (R.S.H., T.G.D., S.-P.O., M.G.);
and Department of Pharmacology and Toxicology, Medical Faculty, Dresden University of Technology, Dresden, Germany
(T.C., U.R.)
Received June 21, 2005; accepted September 28, 2005
ABSTRACT
The cardiac action potential is generated by a concerted action value for HERG channel activation was 10.5 ␮M. These results
of different ion channels and transporters. Dysfunction of any of were reproduced on HERG channels expressed in mammalian
these membrane proteins can give rise to cardiac arrhythmias, human embryonic kidney 293 cells. In guinea pig cardiomyo-
which is particularly true for the repolarizing potassium chan- cytes, studied by patch clamp, application of 10 ␮M NS1643
nels. We suggest that an increased repolarization current could activated IKr and significantly decreased the action potential
be a new antiarrhythmic principle, because it possibly would duration to 65% of the control values. The effect could be
attenuate afterdepolarizations, ischemic leak currents, and re- reverted by application of the specific HERG channel inhibitor
entry phenomena. Repolarization of the cardiac myocytes is 4Ј-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidinyl]carbonyl]-
crucially dependent on the late rapid delayed rectifier current methanesulfonanilide (E-4031) at 100 nM. Application of
(IKr) conducted by ether-a-go-go-related gene (ERG) potassium NS1643 also resulted in a prolonged postrepolarization refrac-
channels. We have developed the diphenylurea compound 1,3- tory time. Finally, cardiomyocytes exposed to NS1643 resisted
bis-(2-hydroxy-5-trifluoromethyl-phenyl)-urea (NS1643) and reactivation by small depolarizing currents mimicking early af-
tested whether this small organic molecule could increase the terdepolarizations. In conclusion, HERG channel activation by
activity of human ERG (HERG) channels expressed heterolo- small molecules such as NS1643 increases the repolarization
gously. In Xenopus laevis oocytes, NS1643 increased both reserve and presents an interesting new antiarrhythmic ap-
steady-state and tail current at all voltages tested. The EC50 proach.
The action potential initiates and controls the contraction delayed rectifier current (IKs) (Snyders, 1999). Most of the ion
of cardiac cells. The shape and duration of the action poten-
tial are the result of an ordered sequence of changes in
membrane permeability to specific ions. The action potential
duration (APD) and refractoriness are especially sensitive to
the membrane permeability to potassium ions. Voltage-gated
potassium channels are activated at different stages of the
action potential, including the early transient outward cur-
rent (Ito) and ultrarapid delayed rectifier current (IKur) as
well as the late rapid delayed rectifier current (IKr) and slow
channels responsible for these currents will undergo inacti-
vation during sustained depolarization. Release from inacti-
vation is seen upon repolarization to the resting membrane
potential. Inactivation is especially pronounced and fast for
IKr current. This implies that IKr current contribution is
almost negligible during the action potential plateau phase
but very prominent during repolarization and the start of the
diastolic interval. The large IKr current at the early diastolic
interval is a consequence of the slow deactivation kinetics of
this channel (Hancox et al., 1998). IKr and IKs are well char-
acterized and can be separated based on their sensitivity
toward organic and inorganic blockers (Sanguinetti and
Jurkiewicz, 1990). It is now widely accepted that the IKr
Article, publication date, and citation information can be found at
http://molpharm.aspetjournals.org.
doi:10.1124/mol.105.015859.
ABBREVIATIONS: APD, action potential duration; HERG, human ether-a-go-go-related gene; NS1643, 1,3-bis-(2-hydroxy-5-trifluoromethyl-
phenyl)-urea; HEK, human embryonic kidney; I-V, current-voltage; APD90, action potential duration at 90% repolarization; EAD, early afterdepo-
larization; E-4031, 4Ј-[[1-[2-(6-methyl-2-pyridyl)ethyl]-4-piperidinyl]carbonyl]methanesulfonanilide; RPR260243, (3R,4R)-4-[3-(6-methoxyquinolin-
4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid.
266

HERG Channel Activation
267
current is mediated by the potassium channel encoded by the
human ether-a-go-go-related gene (HERG) (Sanguinetti et
al., 1995), whereas IKs is constituted by the KCNQ1 ␣-sub-
units together with KCNE1 (MinK) ␤-subunits (Barhanin et
al., 1996; Sanguinetti et al., 1996). It has been suggested that
either KCNE1 or KCNE2 (MiRP1) ␤-subunits are obligatory
components of native IKr (McDonald et al., 1997). However,
this remains controversial (McDonald et al., 1997; Abbott et
al., 1999; Weerapura et al., 2002). Together, IKs and IKr
currents define the late repolarization phase of the cardiac
action potential and are mainly responsible for terminating
the plateau phase.
Most arrhythmias originate from disturbances in the func-
tion of ion channels that generate the normal action poten-
tials. Class III antiarrhythmic drugs block cardiac potassium
channels and prolong the duration of the action potential. As
a result, a longer effective refractory period is obtained, and
the likelihood of re-entry is diminished. Antiarrhythmic
drugs that compromise HERG channel function without ef-
fect on Ca2ϩ channels such as dofetilide or d-sotalol introduce
a risk of proarrhythmic events because they reduce the re-
polarization reserve and may therefore lead to increased
susceptibility to ventricular arrhythmias and eventually to
sudden cardiac death. Pharmacological block of HERG chan-
nels or loss-of-function mutations prolongs APD, which is
reflected on the ECG as a longer QT interval. Ventricular
action potential prolongation can develop into torsade de
pointes arrhythmia and ventricular fibrillation (Monahan et
al., 1990; Sanguinetti and Jurkiewicz, 1990; Metzger and
Friedman, 1993; Pohjola-Sintonen et al., 1993; Woosley et al.,
1993).
In a patent application submitted in 2003, we reported the
first examples of small molecule HERG channel activators
(Olesen et al., 2005). In the meantime, another such com-
pound has been shown to open HERG channels, shorten the
QT interval, and increase T wave amplitude (Kang et al.,
2005).
In the present work, we have applied a new pharmacolog-
ical approach to this potential antiarrhythmic treatment by
identifying the bis-phenol NS1643 as a small molecule acti-
vator of the HERG channel. Our objective was to thoroughly
examine the effect of this agent on heterologously expressed
HERG channels and to test whether the drug has any effect
on action potentials generated by native cardiomyocytes. The
pharmacological impact of this compound supports the idea
of HERG channel activation as a new antiarrhythmic ap-
proach.
Materials and Methods
Chemical Synthesis
NS1643 was synthesized at NeuroSearch A/S (Ballerup, Den-
mark). The symmetrical bis-phenol NS1643 was synthesized in two
steps from 2-methoxy-5-trifluoromethylaniline as shown in Fig. 1.
Reaction of two equivalents of the aniline with phosgene provided
the urea (Fig. 1), which was treated with boron bromide in dichlo-
romethane to furnish NS1643 in 80% overall yield.
Molecular Biology
cDNA encoding HERG (KCNH2, Kv11.1) channels was introduced
into the custom-made vector pXOOM, which is optimized for expres-
sion in both Xenopus laevis oocytes and mammalian cells (Jespersen
et al., 2002). cRNA preparation and capping were performed by in
vitro transcription using the mCAP mRNA capping kit (Stratagene,
La Jolla, CA) or T7 mMessage machine kit (Ambion, Austin, TX)
according to the manufacturers’ instructions. mRNA was phenol/
chloroform extracted, ethanol precipitated, and dissolved in Tris-
EDTA buffer to approximate concentrations of 1 ␮g/␮l. For proof of
purity and integrity, mRNA was inspected by gel electrophoresis,
and concentrations were determined photometrically. mRNA was
stored at Ϫ80°C until injection.
Opening of HERG channels could thus present a novel
antiarryhythmic principle, and several genetic studies sup-
port this notion. It has been demonstrated that increasing IKr
by overexpression of HERG, either by adenoviral transfer in
rabbit (Nuss et al., 1999) and guinea pig (Hoppe et al., 2001)
or by transgenic modification in mouse (Royer et al., 2005),
significantly shortens APD, increases the refractory period of
cardiac tissue, and suppresses electrical alternans in dog
(Hua et al., 2004). These are all indications that point toward
an increase of the HERG current as a beneficial antiarrhyth-
mic approach.
Fig. 1. Synthesis of NS1643.

268
Hansen et al.
Expression in X. laevis Oocytes and Mammalian HEK293
Cells
activity was measured in Kulori solution (90 mM NaCl, 1 mM KCl,
1 mM MgCl2, 1 mM CaCl2, and 5 mM HEPES, pH adjusted to 7.4
with NaOH). The exact voltage protocols are indicated in respective
figures. All experiments were performed at room temperature. The
condition of each single oocyte was controlled before measurements
by recording membrane potentials. Only oocytes with membrane
potentials below Ϫ30 mV were used for current recordings.
X. laevis surgery and oocyte treatment were done as described
previously (Grunnet et al., 2001). Oocytes were collected under an-
esthesia (2 g/l tricain; catalog no. A-5040; Sigma-Aldrich, St. Louis,
MO) according to guidelines approved by the Danish National Com-
mittee for Animal Studies. Before injection of 50 nl of mRNA (ap-
proximately 50 ng), oocytes were kept for 24 h at 19°C in Kulori
solution consisting of 90 mM NaCl, 1 mM KCl, 1 mM MgCl2, 1 mM
CaCl2, and 5 mM HEPES, pH 7.4 with NaOH. Injection of mRNA
was accomplished using a Nanoject microinjector from Drummond
Scientific (Broomall, PA). Oocytes were kept at 19°C for 2 to 5 days
before measurements were performed.
HEK293 Cells. All experiments were performed in whole-cell
configuration, voltage-clamp mode at room temperature with an
EPC-9 amplifier (HEKA, Lambrecht/Pfalz, Germany). Pipettes were
pulled from thin-walled borosilicate glass (ModelOhm, Copenhagen,
Denmark) and had a resistance between 1.5 and 2.5 M⍀. A custom-
made perfusion chamber (volume, 15 ␮l) with a fixed AgCl/Ag pellet
electrode was mounted on the stage of an inverted microscope. A
coverslip with HERG-transfected HEK293 cells was transferred to
the perfusion chamber and superfused with physiological solution
(low Kϩ) consisting of 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 1 mM
MgCl2, and 10 mM HEPES, pH 7.4 with NaOH. Pipettes were filled
with solution consisting of 144 mM KCl, 10 mM EGTA, 10 mM
HEPES, and 4 mM ATP, pH 7.2 with KOH. CaCl2 and MgCl2 were
added in concentrations calculated (EqCal; BioSoft, Cambridge, UK)
to give a free Mg2ϩ concentration of 1 mM and a free Ca2ϩ concen-
tration of 100 nM. No zero current or leak current subtraction was
performed during the experiments. Cell capacitance and series re-
sistance were updated before each pulse application. Series resis-
tance values were between 2.5 and 10.0 M⍀, and only experiments
where the resistance remained constant during the experiments
were analyzed. Current signals were low-pass filtered at 3 kHz and
acquired using PULSE software (HEKA).
HERG/MinK Expression in HEK293 Cells
HEK293 cells stably transfected with the HERG/MinK complex
were used for patch-clamp experiments. The cells were cultured in
Dulbecco’s modified Eagle’s medium with ultraglutamin 1 (Cambrex
Bio Science Walkersville, Inc., Walkersville, MD) supplemented with
10% fetal calf serum at 37°C in 5% CO2.
Isolation of Single Ventricular Cardiomyocytes
Ventricular cardiomyocytes were isolated using a method slightly
modified from that of Mitra and Morad (1985). Perfusion velocity was
10 to 15 ml/min for all solutions, and all solutions were heated to
37°C and filtered before use. Guinea pigs were anesthetized with
intraperitoneal injection of pentobarbital (50–75 mg/kg). In addition,
1 ml/kg heparin (1000 IU/ml) was injected in vena femoralis. Respi-
ration was maintained by artificial ventilation through a cannula in
trachea (volume, 12 ml/kg; rate, 60 strokes/min). Upon thoracotomy,
a perfusion cannula was inserted and fixed in aorta for retrograde
perfusion in a simplified Langendorff setup. To release intracardiac
pressure, a small incision was made in the pulmonary artery. The
heart was surgically removed from the thorax and perfused for 5 min
with oxygenated (100% O2) Tyrode’s solution (with Ca2ϩ) consisting
of 135 mM NaCl, 4 mM KCl, 1 mM MgCl2, 0.33 mM NaH2PO4, 10
mM HEPES, 10 mM glucose, and 2 mM CaCl2, pH 7.4 with NaOH.
This was followed by 5-min perfusion with oxygenated (100% O2)
no-Ca2ϩ Tyrode’s solution. Perfusion was continued for 5 min with
an oxygenated potassium gluconate solution consisting of 120 mM
potassium gluconate, 20 mM NaCl, 1 mM MgCl2, 10 mM HEPES,
and 10 mM glucose, pH 7.4 with KOH. Glucose and potassium
gluconate (28.10 g/l) were added just before use. Finally, enzymatic
digestion was performed by perfusion with oxygenated (100% O2)
potassium gluconate solution containing 0.5 mg/ml collagenase (type
CLS-2; catalog no. LS004176, Medinova Scientific A/S, Glostrup,
Denmark) and 50 ␮M CaCl2. The first 25 ml of this solution was
discarded. The remaining solution was continuously recycled until
the heart seemed swelled, and glassy and perfusion velocity in-
creased (typically obtained within 7–10 min). Ventricular muscles
were minced with scissors and dispersed with gentle agitation in
oxygenated (100% O2) potassium gluconate buffer containing colla-
genase and CaCl2. Cells were filtered through an 80-mesh metal
grid, centrifuged 400 rpm for 2 min, and gently resuspended in
potassium gluconate solution without collagenase and CaCl2. Cells
were stored at room temperature until use.
Native Cardiomyocytes. Cells in suspension were transferred to
coverslips and left for 15 min before recordings. Before performing
patch-clamp experiments, cells were transferred to the perfusion
chamber, and superfused for 5 min with potassium gluconate solu-
tion consisting of 120 mM potassium gluconate, 20 mM NaCl, 1 mM
MgCl2, 10 mM HEPES, and 10 mM glucose, pH 7.4 with KOH, before
changing to Tyrode’s solution consisting of 135 mM NaCl, 4 mM KCl,
1 mM MgCl2, 0.33 mM NaH2PO4, 10 mM HEPES, 10 mM glucose,
and 2 mM CaCl2, pH 7.4 with NaOH. The drugs were also added in
this solution. Pipettes were filled with solutions identical to the one
used for HEK293. Pipettes had a resistance between 1.5 and 2.5 M⍀.
When the on-cell configuration was established, cells were held in
the current-clamp mode, and current was injected until the whole-
cell configuration was obtained. Cells with a measured membrane
potential that deviated more than Ϯ15 mV from Ϫ80 mV were
discarded. At the outset, current steps (lasting 2 ms) from 1000 to
5000 pA were applied in 500-pA increments. Having identified an
appropriate amount of current injection necessary for evoking action
potentials, all further action potentials were initiated by a pulse of
1.2 times the rheobase value. To mimic early and late afterdepolar-
izations, repetitive current injections of approximately 0.5 times
rheobase values were applied at an appropriate time after evocation
of the first action potential. To assess the refractory period, a se-
quence of premature currents was injected as close to the repolariz-
ing action potential as possible without prolonging action potential
duration. Voltage signals were low-pass filtered at 3 kHz and ac-
quired using PULSE software (HEKA).
Native Cardiomyocytes for Recordings of Ca2؉ and Na؉
Current. Currents were measured with the single electrode voltage-
clamp method as described in detail previously (Christ et al., 2005).
The pipette solution had the following composition: 90 mM cesium
Electrophysiological Recordings
Oocytes. Current through expressed HERG channels was moni-
tored using a two-electrode voltage-clamp amplifier (CA-1B; Dagan, methanesulfonate, 20 mM CsCl, 10 mM HEPES, 4 mM MgATP, 0.4
Minneapolis, MN). Electrodes were pulled from borosilicate glass mM Tris-GTP, 10 mM EGTA, and 3 mM CaCl2, with calculated free
capillaries on a horizontal patch-electrode puller (DMZ universal Ca2ϩ concentration of ϳ60 nM, pH 7.2 (EqCal; Biosoft). Ca2ϩ cur-
puller; Zeitz Instruments, Munich, Germany) and had tip resistance rents were measured with Naϩ-free superfusion solution consisting
between 0.3 and 2.5 M⍀ when filled with 1 M KCl. During the
experiments, oocytes were placed in a small chamber (volume, 200
of 120 mM tetraethylammonium chloride, 10 mM CsCl, 10 mM
HEPES, 2 mM CaCl2, 1 mM MgCl2, and 20 mM glucose, pH 7.4
␮l) connected to a continuous flow system (flow, 3 ml/min). HERG (adjusted with CsOH). L-type calcium current (ICa,L) was measured
channels were activated by membrane depolarization and channel from a holding potential of Ϫ80 mV with test steps (200 ms) between

HERG Channel Activation
269
Ϫ70 and ϩ65 mV in 5-mV increments at 37°C. For measuring so-
dium current (INa), 5 mM NaCl was added to the superfusion solu-
tion, and CaCl2 was reduced to 0.5 mM. Contaminating ICa,L was
blocked by nisoldipine (1 ␮M). INa was measured at room tempera-
ture from a holding potential of Ϫ100 mV, with test steps (100 ms)
between Ϫ80 and ϩ5 mV in 5-mV increments. ICa,L and INa ampli-
tudes were determined as the difference between peak inward cur-
rent and current at the end of the depolarizing step. A system for
rapid solution changes allowed application of drugs in the vicinity of
the cells (Cell Micro Controls, Virginia Beach, VA; ALA Scientific
Instruments, Long Island, NY).
Drugs and Solutions
Unless otherwise mentioned, all chemicals were of analytical
grade and were obtained from Sigma-Aldrich. Nisoldipine was a gift
of Baver AG (Wuppertal, Germany). Drugs were dissolved in di-
methyl sulfoxide as concentrated stock solutions and diluted directly
into the superfusion solution to yield the final concentration. Di-
methyl sulfoxide concentration never exceeded 0.1% in final solu-
tions. At this concentration, no influence on any measurements was
observed (data not shown).
Analysis of Data
Data analysis and drawings were performed using IGOR software
(Wavemetrics, Lake Oswego, OR) or Prism software (GraphPad Soft-
ware Inc., San Diego, CA). All deviations of calculated mean aver-
ages are given as S.E.M. values. Significance was calculated as
paired t test or by analysis of variance.
Inactivation data were calculated as normalized peak current data
as a function of the previous membrane potential. Data were nor-
malized and fitted to a Boltzmann sigmoidal function: I ϭ Imax/(1 ϩ
exp[(Vt Ϫ V50)/k]), where I is the current, V50 is the voltage required
for half activation, Vt is the test membrane potential, and k is the
slope factor. Deactivation was calculated by fitting to the double
exponential function: Itail ϭ K0 ϩ Kfast ϫ exp[Ϫ(t/␶fast)] ϩ Kslow
exp(t/␶slow), where t is time in seconds and fast and slow deactivation
constants are determined by ␶ and ␶slow, respectively.
ϫ
fast
Results
A number of drugs and chemical compounds inhibit cur-
rent flow through HERG potassium channels, and it is gen-
erally agreed that these compounds may potentially evoke
lethal cardiac arrhythmias. It has recently proven possible to
synthesize compounds having the opposite effect (i.e., com-
pounds that increase HERG channel activity). Figure 1
shows the structure of such a compound, named NS1643, and
the present work was devoted to a thorough in vitro charac- oocytes is increased in the presence of NS1643. HERG channel activation
terization of its ability to open HERG channels.
To obtain information about the impact of 30 ␮M NS1643
Fig. 2. The current amplitude of HERG channels expressed in X. laevis
was obtained by application of a step protocol from Ϫ80 to ϩ60 mV with
20-mV increments between steps. Tail current was recorded at Ϫ60 mV.
Representative traces are shown in A and B. The steady-state current
on HERG channel activity, heterologous expression of HERG
in X. laevis oocytes was performed. Channel activity was
initiated by different voltage-clamp protocols. Figure 2A is a
representative example of a HERG channel activated by 2-s
voltage steps from a holding potential of Ϫ80 mV to poten-
tials ranging from Ϫ80 to ϩ60 mV. Increment between steps
was ϩ20 mV, and tail current was recorded at Ϫ60 mV for
5 s. As expected, HERG channels responded to this protocol
with a voltage-dependent activation followed by strong inac-
tivation. As a consequence of the inactivation, the I-V curve
recorded during the step protocol from Ϫ80 to ϩ60 mV had a
bell-shaped appearance with maximal amplitude at 0 mV
(Fig. 2C). Release from inactivation is revealed by the instant
large tail current observed upon repolarization to Ϫ60 mV.
revealed strong inactivation as seen by the bell-shaped I-V curve (open
squares; C), whereas a saturation in tail current was observed at poten-
tials more positive than 10 mV (closed circles; E). Application of 30 ␮M
NS1643 gave a robust increase in current amplitude in both steady-state
current (D) and tail current (F). Inactivation properties were addressed
by a voltage protocol starting at ϩ20 mV for 1 s, followed by 10-ms
hyperpolarizing steps from Ϫ150 to ϩ20 mV, before again clamping the
potential at ϩ20 mV for 1 s. Data were normalized and fitted to a
Boltzmann function (G and H). Analysis of inactivation data in control
oocytes revealed a V50 of Ϫ72.7 Ϯ 2 mV and a slope factor of 15.9 Ϯ 2. For
oocytes exposed to NS1643, V50 was Ϫ63.7 Ϯ 3 mV and the slope factor
was 16.8 Ϯ 3 mV. The difference in V50 is significant (p ϭ 0.02). For all
summarized experiments, n ϭ 6. I and J show the effect of NS1643 on
HEK293 cells stably expressing the HERG channel and the KCNE1
subunit. Voltage-clamp recordings were performed by applying the pro-
tocol outlined in A. When stable control currents were obtained, 10 ␮M
NS1643 was added to the cells. Application of the drug lead to a 45.1 Ϯ
10.1% increase of the tail peak current and a 20 Ϯ 11.9% increment of the
Summarized tail current data are shown in Fig. 2E. When steady-state current measured at 0 mV relative to the control (n ϭ 3).

270
Hansen et al.
HERG-expressing oocytes were challenged by similar voltage from Ϫ130 to ϩ40 mV are depicted in Fig. 3, E and F. No
protocols in the presence of 30 ␮M NS1643, an increase in significant difference in deactivation kinetics could be ob-
current was obvious in both steady-state current and tail served after application of NS1643, even though there was a
current (Fig. 2, B, D, and F). Bolzmann fits of activation tendency toward decreasing ␶fast and ␶slow values in the pres-
revealed a half-maximal activation of Ϫ10.0 Ϯ 2 mV for ence of NS1643.
control experiments and Ϫ10.0 Ϯ 3 mV for recordings in the
In an attempt to reveal the time course for HERG activa-
presence of NS1643. Inactivation properties in the presence tion by NS1643, oocytes were continuously challenged by a
and absence of NS1643 were investigated by a complete voltage protocol starting at ϩ20 mV for 1 s followed by 3 s at
activation and inactivation of the channels by clamping the Ϫ60 mV. Cells were clamped for 3 s at Ϫ80 mV between
membrane potential to ϩ20 mV for 1 s. This was followed by steps. Figure 4A demonstrates how HERG channels re-
brief (10-ms) hyperpolarized steps from Ϫ150 to ϩ20 mV,
before the potential was again clamped for 1 s at ϩ20 mV.
The 10-ms hyperpolarized step results in a release from
inactivation and is sufficiently short to prevent initiation of
deactivation. From this protocol, the voltage dependence of
recovery from inactivation was determined by plotting the
peak current recorded, at the second clamp to ϩ20 mV, as
function of the previous potential (Fig. 2, G and H). Data
were normalized and fitted to a Boltzmann function. In con-
trol oocytes, V50 was Ϫ72.7 Ϯ 2 mV and the slope factor was
15.9 Ϯ 2 mV. For oocytes exposed to NS1643, V50 was
Ϫ63.7 Ϯ 3 mV and the slope factor was 16.8 Ϯ 3 mV. Sum-
marized data represent n ϭ 6. Because it has been reported
that compounds can have a different profile when tested in
different expression systems, experiments were also con-
ducted with HERG channels expressed in a mammalian cell
system (Fig. 2, I and J). The effect of NS1643 as a HERG
channel activator was investigated in HEK293 cells stably
expressing HERG channels together with the ␤-subunit
KCNE1 (MinK). In initial experiments, application of 30 ␮M
NS1643 was performed; however, long-lasting whole-cell re-
cordings were difficult to obtain because seals were unstable
in the presence of this compound concentration. Experiments
were therefore performed at lower compound concentration
than was applied in oocyte experiments. When stable control
currents were obtained, 10 ␮M NS1643 was added to the
bath. Application of the drug to the cells led to an increase of
the peak tail current of 45.1 Ϯ 10.1% (n ϭ 3) (Fig. 2J). To
confirm expression of HERG channels and to determine the
magnitude of leak, current experiments were completed by
adding the specific HERG channel blocker E-4031 at 100 nM,
which in mammalian cells is sufficient to obtain a complete
inhibition of HERG current (data not shown).
The effect of NS1643 on the tail current and deactivation
kinetics at different voltages was monitored by a protocol
whereby oocytes were fully activated and inactivated by
clamping to ϩ40 mV for 1 s, followed by steps from Ϫ130 to
ϩ40 mV, lasting for 4 s. Increment between steps was 10 mV.
Between steps, oocytes were clamped for 3 s at Ϫ80 mV.
Representative results are depicted in Fig. 3. Fast activation
Fig. 3. HERG tail current and deactivation kinetics in the presence of
and inactivation were similar both in the presence and ab-
sence of 30 ␮M NS1643. In contrast, an increase in peak tail
current amplitude was observed in the presence of NS1643
compared with controls. In addition, a rightward shift was
observed for maximal peak current amplitude in the pres-
ence of NS1643. In control situations, the largest tail current
could be recorded at Ϫ50 mV, whereas results obtained in the
presence of NS1643 revealed a maximal tail current at Ϫ40
mV. Summarized data are plotted as I-V curves (n ϭ 6). The
deactivation kinetics for tail currents was calculated by fit-
ting to a double exponential function. Summarized numbers
NS1643. HERG channels expressed in X. laevis oocytes were activated
and completely inactivated by holding at ϩ40 mV for 1 s. Voltage depen-
dence of release from inactivation, in the presence and absence of
NS1643, was studied by a tail protocol stepped from Ϫ130 to ϩ40 mV.
Current recorded at the peak of the tail resulted in a bell-shaped I-V
curve with a maximum current at Ϫ50 mV for control experiments and
Ϫ40 mV for recording in the presence of NS1643 (C and D). Application
of 30 ␮M NS1643 gave approximately a current increase of 60% at all
voltage potentials (C and D). Deactivation was calculated by fitting to a
double exponential function. Summarized numbers for ␶
and ␶
fast
slow
values at preceding membrane potentials from Ϫ130 to ϩ40 mV are
depicted in E and F. No significant difference in deactivation kinetics
could be observed after application of NS1643, even though there was a
tendency toward decreasing ␶
and ␶
values in the presence of
fast
slow
for ␶
and ␶
values at preceding membrane potentials NS1643 (n ϭ 6 for summarized data).
fast
slow

HERG Channel Activation
271
sponded to a single pulse of this protocol. Current recorded at both high specificity and affinity when applied in mamma-
ϩ20 mV was slightly increased in the presence of NS1643 lian expression systems with complete block observed with
compared with control. Tail currents recorded at Ϫ60 mV in less than 100 nM E-4031. The affinity for HERG channels
the presence of NS1643 revealed a robust increase in current expressed in X. laevis oocytes, however is, severely compro-
amplitude during the entire time course of the deactivation. mised. E-4031 (100 nM) was not able to reduce HERG cur-
An online analysis of this protocol repeated continuously is rent, and a 100-fold increase in concentration only reduced
depicted in Fig. 4B. Peak tail current as a function of time the current to 35% of control value. Such change in affinity
reveals how addition of 30 ␮M NS1643 increases HERG of E-4031 between expression systems has been reported
current approximately 100% within a time scale of roughly previously (Sanguinetti et al., 1995). The EC50 value for
200 s. Washing for 400 s only reduced the current level NS1643 activation of HERG channels was calculated to
approximately 50%. To confirm specificity, the experiment 10.5 Ϯ 1.5 ␮M.
was finalized by addition of HERG channel inhibitor halo-
Because KCNQ1 channels and HERG channels are both
peridol (10 ␮M). A rapid decrease in current amplitude that major players in repolarization of the cardiac action potential
could be reverted by washing was observed. In noninjected as part of the IKs and IKr complexes, respectively, it is logical
oocytes, the applied voltage protocol only resulted in insig- to investigate whether NS1643 could have an impact on
nificant endogenous current activation of less than 200 nA in KCNQ1 current. For this purpose, oocytes expressing
maximal amplitude.
KCNQ1 channels were activated by voltage ramps from
The protocol applied in time-course experiments was also Ϫ100 to ϩ60 mV in 20-mV increments followed by tail re-
used in defining the EC50 value for HERG activation by cordings at Ϫ30 mV. As seen in Fig. 6A, characteristic slowly
NS1643. These studies were conducted with oocytes exposed activating KCNQ1 currents were obtained. Application of 30
to only a single concentration of NS1643 in the range from 1 ␮M NS1643 resulted in a reduction in current amplitude
to 300 ␮M. Results were monitored as increase in peak tail without any obvious effect on kinetic parameters (Fig. 6B).
current. Data are summarized in Fig. 5A. As seen, saturation On average, application of 30 ␮M NS1643 reduced KCNQ1
in increment of HERG current was observed at concentra- current to 64.7 Ϯ 9% of control level (n ϭ 5). Because the
tions equal to or above 100 ␮M. Specificity of current was cardiac IKs current is composed of a channel complex contain-
confirmed by application of 10 ␮M haloperidol or the more ing both KCNQ1 and KCNE1, the ability of NS1643 to inhibit
specific HERG channel inhibitor E-4031. This compound has this current was also investigated. As can be seen in Fig. 6, D
Fig. 4. Time course of HERG channel
activation by NS1643. HERG channel
expressed in X. laevis oocytes were
repeatedly activated by clamping 1 s
at ϩ20 mV followed by 3 s at Ϫ60 mV.
Between steps, cells were kept at Ϫ80
mV for 3 s. Current traces for a single
step in the presence and absence of 30
␮M NS1643 is demonstrated in A. The
time course for current recorded at
the peak of the tail current (indicated
by arrow), is depicted in B. From a
stable level of approximately 4 ␮A,
application of 30 ␮M NS1643 gave a
current increase to approximately 8
␮A. The effect of NS1643 could be
partly reversed by washing. Specific-
ity of HERG current was confirmed by
application of 10 ␮M haloperidol.
Fig. 5. Concentration-response relationship of NS1643. Oocytes expressing HERG channels were activated by a continuously step protocol from ϩ10
to Ϫ60 mV, and peak tail current was recorded in the presence of different concentrations of NS1643. Only a single concentration of compound was
applied to each single oocyte. HERG channel activity was in addition measured in the presence of the two HERG channel inhibitors, haloperidol (10
␮M) and E-4031 (100 nM and 10 ␮M) (A). Increase in HERG channel activity as a function of the concentration of NS1643 was calculated (EC50 of
10.5 Ϯ 1. 5 ␮M) (B). Between five and eight single oocytes were tested at every concentration of NS1643.

272
Hansen et al.
to F, a pronounced current inhibition of KCNQ1/KCNE1 (representing IKur) were studied by expression of the human
current was observed. On average, application 30 ␮M cloned genes in X. laevis oocytes. Channel activation was
NS1643 reduced KCNQ1/KCNE1 current to 34.5 Ϯ 28.4% obtained by voltage step protocols from Ϫ80 to ϩ40 mV.
of control level (n ϭ 5). To further investigate the effect of NS1643 (30 ␮M) inhibited Kv4.3 current to 84.6 Ϯ 8% of
NS1643 by simultaneously application to both KCNQ1 and control levels when current was recorded at ϩ40 mV (Fig. 7,
HERG channels, coexpression studies were performed in X. A–C; n ϭ 4). In contrast, no effect was observed on Kv1.5
laevis oocytes. Channel activation was achieved by a voltage currents (Fig. 7, D–F; n ϭ 3). The effect of NS1643 on ICa,L
protocol starting with 3 s at Ϫ80 mV, followed by 1 s at ϩ10 and T-type calcium currents (ICa,T) as well as INa was exam-
mV and finalized with 3 s at Ϫ60 mV. With this protocol, ined in experiments performed with native guinea pig ven-
KCNQ1 contribution to the summarized current will be most tricular cardiomyocytes. Five minutes after establishing ac-
prominent at ϩ10 mV because of the strong inactivation of cess by whole-cell patch clamping, the cells were exposed to
HERG current at this potential, whereas the HERG current 10 ␮M NS1643. ICa,L exhibited a substantial and variable
contribution will have a large impact at Ϫ60 mV as a conse- rundown over time when exposed the drug; however, this
quence of the release from inactivation. When NS1643 was decline was not different compared with the spontaneous
applied to coexpressing oocytes, a current reduction was ob- rundown in time-matched controls (i.e., from 12.9 Ϯ 1.5 to
served at ϩ10 mV, whereas the tail current recorded at Ϫ60 10.8 Ϯ 1.4 pA/pF; n ϭ 11). ICa,L density was 13.1 Ϯ 2.1 pA/pF
was increased (Fig. 6G). The corresponding on-line analysis before and 10.7 Ϯ 2.1 pA/pF after drug exposure (P Ͻ 0.05;
recorded at ϩ10 and Ϫ60 mV, respectively, is shown in Fig. n ϭ 13). Moreover, NS1643 also did not affect ICa,T (Fig. 7, G
6, H and I. These results demonstrate that the overall effect and H). Furthermore, we examined the effect of NS1643 on
of NS1643 on action potentials recorded from native cells is native sodium currents. INa was stable over time, and the
likely to depend on the relative contribution of HERG and current density 2 min after exposure to 10 ␮M NS1643 was
KCNQ1 to the repolarizing phase 3 current.
The selectivity of NS1643 toward other important cardiac versus 25.3 Ϯ 4.1 pA/pF; n ϭ 7) (Fig. 7, I and J).
potassium currents was also characterized. Kv4.3 potassium Because NS1643 was observed to activate HERG in both X.
not different from the respective control values (25.8 Ϯ 3.9
channels (representing Ito) and Kv1.5 potassium channels laevis oocytes and mammalian expression systems, it was
Fig. 6. NS1643 inhibits KCNQ1 current. Oocytes
expressing KCNQ1 channels were activated by step
protocols from Ϫ100 to ϩ60 mV. Current activity was
measured in the absence (A) or in the presence (B) of
30 ␮M NS1643. I-V curves are summarized in C.
Using the same voltage protocol the effect of NS1643
on oocytes expressing both KCNQ1 and KCNE1 was
addressed (D and E). I-V curves are summarized in F.
Dual effect of NS1643 on HERG and KCNQ1 current
was investigated by coexpressing both channel types
in oocytes. Current was activated by a repeated step
protocol from ϩ10 to Ϫ60 mV (G). Current trace in
the absence of NS1643 is labeled in black, whereas
the current trace recorded in the presence of 30 ␮M
NS1643 is labeled in gray. NS1643 inhibited current
at ϩ10 mV where KCNQ1 contributes the majority of
the activity. In contrast, NS1643 amplified current
recorded at Ϫ60 mV where HERG channels are
mostly active. The activity recorded at ϩ10 mV is
demonstrated in H (open squares), whereas current
recorded at Ϫ60 mV is depicted in I (closed circles).

HERG Channel Activation
273
assumed that the drug would also affect the IKr of native the shape of the action potential was not altered. From these
cardiomyocytes. To test this hypothesis, a series of patch- results, we concluded that the IKr of the native cardiomyo-
clamps studies were performed on isolated guinea pig cardi- cytes was activated by NS1643, resulting in a decrease in
omyocytes. Cells were held in current-clamp mode and stim- action potential duration, without any obvious effect on other
ulated for 2 ms with 1 to 5 nA (1.2 times rheobase value) to ion channels involved in the action potential. Moreover, it
induce action potentials. Upon application of 10 ␮M NS1643, was observed that the variability of the action potential du-
action potential duration at 90% repolarization (APD90) was ration was much more pronounced in control action poten-
shortened significantly to 66 Ϯ 8% (n ϭ 3) (Fig. 8) relative to tials than in action potentials elicited after exposure to
control. This effect was reversed by application of 100 nM NS1643, indicating that action potentials are more uniform
E-4031 (data not shown). Furthermore, it was observed that in the presence of 10 ␮M NS1643 (data not shown).
Because of the increase in repolarizing reserve, we hypoth-
esized that NS1643 would affect the refractory period of the
guinea pig cardiomyocytes. To test whether this was true,
action potentials were elicited as described above, and a
second current of same magnitude was applied as close to the
fully repolarized action potential as possible. The refractory
period independently of APD was defined as the time span
from repolarization until it was again possible to elicit a new
action potential as described previously (Nuss et al., 1999).
As can be seen from Fig. 9, application of 10 ␮M NS1643
dramatically and significantly (P Ͻ 0.01) altered the refrac-
toriness of the guinea pig cardiomyocyte from 19 Ϯ 9 to 156 Ϯ
36 ms (n ϭ 3). Subsequent application of 100 nM E-4031
resulted in a prolonged action potential and a decrease in the
postrepolarization refractory period, although the second
stimulus was unable to elicit full-size action potentials
(Fig. 9C).
Fig. 7. Specificity of NS1643. The activity of NS1643 toward Kv1.5
(representing IKur) and Kv4.3 (representing Ito) potassium channels was
investigated after expression in oocytes. Currents were evoked by step
protocols from Ϫ80 to ϩ40 mV. Oocytes were clamped at Ϫ80 mV be-
tween steps and tail currents recorded at Ϫ60 mV. As demonstrated in A
to C, NS1643 applied at 30 ␮M had a minor inhibitory effect on Kv4.3
current. The current amplitude was reduced to 84.6 Ϯ 8% of control
levels. No effect on Kv1.5 current was observed after application of 30 ␮M
NS1643 (D–F). The ability of NS1643 to affect L-type voltage-gated Ca2ϩ
channels and cardiac Naϩ channels was addressed using guinea pig
ventricular myocytes. Current-voltage relationship obtained by applica-
tion of the depicted protocol for Ca2ϩ current recorded before and 2 min
after addition of 10 ␮M NS1643 (G). The small shoulder in the I-V curve
between Ϫ45 and Ϫ20 mV represents T-type Ca2ϩ current; the remaining
current (peak at ϩ5 mV) is due to L-type current (Heubach et al., 2000).
Time course of peak ICa,L is shown in H. The current-voltage relationship
for INa using the outlined protocol recorded before and after application of
10 ␮M NS1643 is demonstrated in I and the time course for peak INa in
J. H and J show original traces obtained at times marked by a and b,
respectively. The horizontal bars indicate the time of drug exposure.
Fig. 8. APD measured from acutely isolated ventricular guinea pig car-
diomyocytes. Cells were held in the current-clamp mode and stimulated
to induce action potentials with injection of 120% threshold current at 0.5
Hz. When stable action potentials were obtained, APD was measured at
APD90 (A). Addition of 10 ␮M NS1643 abbreviated APD90 significantly
(P Ͻ 0.01) from 568.21 Ϯ 60.1 to 373.5 Ϯ 10.2 ms (n ϭ 3) (B).

274
Hansen et al.
Early afterdepolarizations (EADs) and triggered activity triggered in the control cells when the premature stimuli
can be caused by reactivation of L-type Ca2ϩ channels and were applied at APD90. This effect was observed in all control
may occur when premature stimuli coincide with an action experiments (n ϭ 3). In contrast, when 10 ␮M NS1643 was
potential in the repolarizing phase. Together with a reduc- applied, the action potential was only marginally affected by
tion of repolarizing current, increase of sodium-calcium ex- the premature sequence at APD90 (Fig. 10, D and E; n ϭ 3),
changed currents, and an increase in late sodium current, indicating that NS1643 stabilizes the action potential as an
premature reactivation of L-type Ca2ϩ current is generally add-on to its prolongation of refractoriness. The effect seen
believed to be one of the cellular mechanisms underlying with NS1643 was completely antagonized by application of
initiation of torsade de pointes and related polymorphic E-4031 (data not shown).
tachycardias (Nattel and Quantz, 1988; Szabo et al., 1995;
Patterson et al., 1997; Burashnikov and Antzelevitch, 1998).
Discussion
L-type Ca2ϩ channels are activated at membrane potentials
around Ϫ30 mV, and because NS1643 was shown to hasten
In the present study, the bis-phenol NS1643 was shown to
repolarization and increase refractoriness, we hypothesized activate cloned HERG channels and to abbreviate action
that application of NS1643 could counteract the effect of potentials recorded from isolated cardiomyocytes. The reduc-
simulated EADs. To test whether this was true, cells were tion of the action potential duration was seen without other
held in current-clamp mode, action potentials were elicited, obvious changes in action potential shape, and the shorten-
and the cell was stimulated at APD90 with a sequence of 10 ing was reversible upon application of the specific HERG
to 20 premature currents at 50% of the stimulation current. blocking compound E-4031. These findings suggest that the
The train of current injections was adjusted so it did not elicit molecular mechanism of action of NS1643 is to primarily
an action potential when applied after the action potential activate the native HERG channels underlying the IKr of the
had fully repolarized (Fig. 10, A and C). As seen on Fig. 10, B cardiomyocytes, as was further supported by the selectivity
and E, a pronounced prolongation of the action potential was data for the compound.
Fig. 9. Influence of NS1643 on the refrac-
toriness of guinea pig cardiomyocytes. Ac-
tion potentials were elicited as described
in Fig. 8, APD was determined, and a
115% threshold current was injected as
close to the repolarizing action potential
as possible, to determine the postrepolar-
ization refractory time. The time period
after repolarization to APD90, in which it
was not possible to elicit a new action
potential, was determined and termed
the postrepolarization refractory time
(A). After application of 10 ␮M NS1643,
this refractory time of the myocytes in-
creased significantly (P
Ͻ 0.01) from
19.3 Ϯ 9.3 to 156.3 Ϯ 36.0 ms (n ϭ 3) (B).
This increment could be completely re-
versed by 100 nM E-4031, which resulted
in a refractory period of 19.0 Ϯ 2.6 ms
(n ϭ 3) (C).

HERG Channel Activation
275
During the course of the cardiac action potential, the inent enough to affect the plateau phase. Another possibility
HERG channels open at membrane depolarizations positive is that the steady-state current of the native HERG channel
to Ϫ40 mV. The channel open state, however, is transient, is not affected as strongly as seen in experiments performed
and the inactivation time constant is very fast (Zhou et al., with cloned channels, which may be due to the presence of
1998; Kiehn et al., 1999). As a consequence of this mecha- native subunits and/or other intracellular modulators such
nism, HERG channels only provide a small steady-state cur- as described by Sanguinetti and Jurkiewicz (1990). Finally,
rent during the action potential plateau phase (phase 2). the compound was also found to block the KCNQ1 current,
When the membrane potential repolarizes, the HERG chan- and blocking of this slowly activating current during the
nels are released from inactivation. As a result, a large peak plateau phase may counteract the activation of the HERG
current with a long-lasting decay is seen, reflecting a slow steady-state current.
deactivation time constant. Exposure to NS1643 increased
Kang et al. (2005) presented the first activator of the
the HERG channel steady state as well as the peak currents HERG channel, RPR260243, which mediated its action by an
in a concentration-dependent manner. Comparison of kinetic extreme slowing of the channel deactivation, whereas the
parameters revealed a rightward shift of V50 for inactivation compound affected neither the steady-state nor the peak tail
of 9 mV in the presence of NS1643 (Fig. 2, G and H). Activa- current. In native cardiomyocytes, 30 ␮M RPR260243 short-
tion of the HERG steady-state current during the plateau ened the action potential duration by 12%, whereas no APD
phase of an action potential would be expected to affect the shortening effect was observed using lower concentrations.
time course of the plateau phase, which was not seen in the The deactivation of the cloned HERG channel was, in con-
present experiments. Several reasons can account for this. trast, slowed down by 10 ␮M RPR260243. In the present
First, increase of the steady-state current may not be prom- work, we showed that 10 ␮M NS1643 increased both the
Fig. 10. Response of cardiomyocytes to simulated
EADs. After an elicited action potential, a train of
10 to 20 50% subthreshold stimuli were applied
either at control situations after full repolarization
(A and C), or, to induce EADs, at APD90 (B and D).
In the absence of NS1643, subthreshold stimuli
applied at APD90 were able to induce EAD events
every time the protocol was applied, and prolonga-
tion of the action potential was continued even
after cessation of current injection (B). With appli-
cation of 10 ␮M NS1643 the premature train of
stimuli was not able trigger any abnormal activity
and application of the drug thus led to a stable
action potential (D). Superimposed traces for sub-
threshold stimuli applied at APD90 in the absence
or presence of NS1643 are shown in E, left. Cur-
rent injection protocol is shown in E, right.

276
Hansen et al.
peak tail current and the steady-state current. At the same nels. We therefore believe that pharmacological com-
concentration, the APD measured in cardiomyocytes was ab- pounds with a HERG opener profile such as NS1643 could
breviated by 34%. These findings suggest that the HERG be beneficial as antiarrhythmic drugs preventing EADs.
peak tail current strongly influences the duration of the
In conclusion, we have described the abilities of the bis-
action potential and represents a repolarization reserve, phenol compound NS1643 to activate cloned and native
whereas an increase in deactivation time does not affect the HERG channels. This compound represents a new investiga-
action potential duration to a similar extent. An alternative tional tool for the study of cloned HERG channels and of IKr
explanation could simply be that the two compounds have in cardiomyocytes. NS1643 increases both the steady-state
different affinity for the HERG channel.
and the peak tail current of the cloned HERG channels, gives
Antiarrhythmic drugs have been developed to specifically a rightward shift in V50 for inactivation, and slows the
increase the action potential duration and thereby the effec- deactivation of the tail current. In the native cardiomyo-
tive refractory period by blocking Kϩ channels. Increase of cytes, the drug abbreviates action potential duration, in-
the refractoriness of the cardiac tissue will tend to break creases the postrepolarization refractoriness, and sup-
re-entry loops and has proven effective in preventing some presses hyperexcitability. The present data support the
atrial arrhythmias (Nattel, 2002). The drugs, however, are concept of use of HERG channel activators as a novel
proarrhythmic in other patients, which is probably caused by antiarrhythmic principle.
their reduction in the repolarization capacity and increased
tendency to early aferdepolarization. The present HERG
channel opener has the opposite effect in increasing the re-
Acknowledgments
We thank Camilla Irlind, Lise Lauenborg, and Inge Hyttel for
excellent technical assistance.
polarizing reserve and reducing hyperexcitability. NS1643
also increases the postrepolarization refractory period.
Whether the compound will increase or decrease the effective
refractory period cannot be determined based on the cellular
studies, because the absolute APD in cultured cardiac myo-
cytes may differ from that measured in tissue and tend to be
slightly longer. The key feature of the HERG channel open-
ers is thus to stabilize the myocytes during repolarization
and the following period and because their mechanism is
very different from that of the class III compounds, they are
not expected to be associated with the same proarrhythmic
potential. In fact, it has been shown that overexpression of
HERG channels in mouse led to a smaller susceptibility to
atrial and ventricular fibrillation (Royer et al., 2005), and it
is very likely that the same may be true for a HERG channel-
activating agent.
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Address correspondence to: Dr. Morten Grunnet, NeuroSearch A/S, Ped-
erstrupvej 93, 2750 Ballerup, Denmark. E-mail: mgr@neurosearch.dk