Atrial fibrillation is one of the most frequent types of ar-
rhythmia in clinical practice. It is reported to double the risk
of deaths due to cardiovascular diseases and to be the major
risk factor for thromboembolism, especially cerebral em-
bolism.1) At present, atrial fibrillation is mainly treated with
class I antiarrhythmic agents such as pilsicainide and fle-
cainide, or class III antiarrhythmic agents such as dofetilide
and amiodarone,2—4) but the major problem with these agents
is that they also affect ventricular excitation and repolariza-
tion. Thus, drugs with atrial selectivity are desired for the
treatment of atrial fibrillation.
NIP-142, (3R*,4S*)-4-cyclopropylamino-3,4-dihydro-2,2-
dimethyl-6-(4-methoxyphenylacethylamino)-7-nitro-2H-1-
benzopyran-3-ol, is a benzopyran derivative with terminating
effects on canine vagal stimulation-induced atrial fibrillation
model and on canine Y-shaped incision-induced atrial flutter
model.5—7) These effects have been attributed to prolongation
of atrial refractory period. The prolongation of the refractory
period and action potential duration (APD) by NIP-142 was
observed in the atrium,5,8) but not in the ventricle.8) This may
indicate that NIP-142 is less likely to disturb ventricular re-
polarization when applied for the treatment of atrial arrhyth-
mia.
Concerning the molecular mechanisms for the action po-
tential prolongation by NIP-142, blocking effects on potas-
sium currents such as the acetylcholine-activated potassium
current (IKACh)
8,9) and the ultrarapid component of the de-
layed rectifier potassium current (IKur)
10) has been reported.
The blocking effect on the rapid component of the delayed
rectifier potassium current (IKr) was observed only at higher
concentrations. The fast component (IKr) and the slow com-
ponent (IKs) of the delayed rectifier potassium current are the
two major currents responsible for atrial repolarization in
species including the guinea-pig and human.11) However, the
effect of NIP-142 on IKs has not yet been reported. In the
present study, we examined the effect of NIP-142 on currents
through expressed potassium channel subunits KCNQ1/
KCNE1, which underlie IKs.
MATERIALS AND METHODS
Preparation of HEK293 Cells Expressing Human
KCNQ1/KCNE1 Channel Currents cDNA fragments for
human KCNQ1 and KCNE1, which encode the two subunits
of the IKs channel, were amplified by polymerase chain reac-
tion from a human heart cDNA library (Takara Shuzo Co.,
Ltd., Kyoto, Japan) with oligonucleotide primers designed
based on the published mouse cDNA sequence of KCNQ1
(GenBank accession number U89364) and KCNE1 (Gen-
Bank accession number NM000219), and assembled to
obtain the full length cDNAs. They were inserted into the
vector pIRES (Clontech, Palo Alto, CA, U.S.A.) to yield a
tricistronic expression vector from which a single mRNA
coding KCNQ1, KCNE1, and the neomycin resistance pro-
tein is transcribed. This vector, together with the vector
pIRES-hrGFP-1a (Stratagene, Garden Grove, CA, U.S.A.),
which encodes the green fluorescence protein (GFP), was in-
troduced into HEK293 cells with lipofectamine (Invitrogen,
Tokyo) and stable transformants were obtained by clone cul-
ture in the presence of G418 (500 mg/ml), a neomycin ana-
log. Stable transformants of HEK293 cells expressing
KCNQ1/KCNE1 channels were obtained as described in our
previous report.9) The cells expressing either channel were
plated on glass coverslips 48 to 72 h before electrophysiolog-
ical experiments.
Electrophysiological Recording of Expressed KCNQ1/
KCNE1 Channel Current Whole-cell voltage clamp ex-
periments were performed with HEK293 cells expressing
KCNQ1/KCNE1 channels in a chamber mounted on the
stage of an inverted microscope. The chamber was perfused
continuously at a flow rate of 1.0 to 2.0 ml/min and the tem-
perature was maintained at 22—25 °C. The external solution
January 2011 153Note
Blocking Effect of NIP-142 on the KCNQ1/KCNE1 Channel Current
Expressed in HEK293 Cells
Iyuki NAMEKATA,a Noriko TSURUOKA,a Yayoi TSUNEOKA,a Tomoyuki MATSUDA,a,b Akira TAKAHARA,a
Yoshio TANAKA,a,c Takeshi SUZUKI,a,d Tetsuo TAKAHASHI,e Naoko IIDA-TANAKA,a, f and
Hikaru TANAKA*,a
a Department of Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; c Department of Chemical
Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; e Department of Biophysical Chemistry, Faculty of
Pharmaceutical Sciences, Toho University; Funabashi, Chiba 274–8510, Japan: b Biological Research Laboratories,
Nissan Chemical Industries, Ltd.; Shiraoka, Saitama 349–0294, Japan: d School of Material Science, Japan Advanced
Institute of Science and Technology; Tatsunokuchi, Ishikawa 923–1292, Japan: and f Department of Food Science, Otsuma
Women’s University; Chiyoda-ku, Tokyo 102–8357, Japan.
Received September 14, 2010; accepted October 29, 2010; published online November 5, 2010
We examined the effect of NIP-142, a benzopyran compound with terminating effect on experimental atrial
arrhythmia, on the KCNQ1/KCNE1 channel, which underlies the slow component of the cardiac delayed recti-
fier potassium channel (IKs). NIP-142, as well as chromanol 293B, showed concentration-dependent blockade of
the current expressed in HEK293 cells; the EC50 value of NIP-142 and chromanol 293B for the inhibition of tail
current was 13.2 mM and 4.9 mM, respectively. These results indicate that NIP-142 has blocking effect on the
KCNQ1/KCNE1 channel current.
Key words NIP-142; KCNQ1; KCNE1; atrial fibrillation
Biol. Pharm. Bull. 34(1) 153—155 (2011)
© 2011 Pharmaceutical Society of Japan∗ To whom correspondence should be addressed. e-mail: htanaka@phar.toho-u.ac.jp
was of the following composition: 128.0 mM NaCl, 20.0
mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 0.33 mM NaH2PO4,
5.0 mM N-(2-hydroxyethyl)piperazine-N�-2-ethanesulfonic
acid (HEPES), 10.0 mM glucose (pH 7.4 with NaOH). The
patch pipette solution was of the following composition:
100 mM KOH, 40 mM KCl, 70 mM aspartic acid, 1 mM MgCl2,
5 mM ATP-K2, 5 mM creatine phosphate-K2, 5 mM HEPES,
10 mM ethylene glycol bis(2-aminoethylether)-N,N,N�,N�-
tetraacetic acid (EGTA) (pH 7.2 with KOH). Pipette tip re-
sistances were 2 to 4 MW when filled with the patch pipette
solution. The KCNQ1/KCNE1 channel current was meas-
ured as the amplitude of the peak tail current on return to a
holding potential of �40 mV from 3 s voltage clamp pulses.
Data acquisition and analyses were performed with the sys-
tem described previously9) including a patch-clamp amplifier
(Axopatch 1D; Axon Instruments, Foster City, CA, U.S.A.),
a personal computer (Prolinea 486; Compaq, Houston, TX,
U.S.A.) and pCLAMP software (Axon).
Drugs and Chemicals NIP-142 was synthesized and
provided by Nissan Chemical Industries (Tokyo, Japan).
NIP-142 was added to the bath solution from a stock solution
(100 mM) in 0.1 M HCl. The final concentration of HCl in the
measuring bath (�0.1 mM) did not affect any of the experi-
mental parameters measured. All other chemicals were com-
mercial products of the highest available grade of quality.
RESULTS
In whole cell voltage clamped HEK293 cells expressing
the KCNQ1/KCNE1 channel, depolarization to membrane
potentials more positive than �10 mV induced voltage- and
time-dependent outward currents. On repolarization to �40
mV, outward tail currents were observed (Figs. 1A, C). The
peak amplitude of the tail current after a depolarizing pulse
to 20 mV was 8.6�0.8 pA/pF (n�10).
Chromanol 293B, at 1 to 30 mM, concentration-depen-
dently reduced the outward currents on depolarization and
the tail currents on repolarization (Figs. 1A, B). The current
density of the tail current after a depolarizing pulse to 20 mV
in the absence and presence of 1, 3, 10 and 30 mM chromanol
293B was 7.5�1.4 pA/pF (n�10), 5.4�0.4 pA/pF (n�4),
4.5�0.9 pA/pF (n�5), 2.2�0.2 pA/pF (n�6) and 0.1�0.2
pA/pF (n�4), respectively. The EC50 value for the inhibition
of tail current on repolarization from 20 mV was 4.9 mM (Fig.
1E).
NIP-142, at 10 and 30 mM, concentration-dependently re-
duced the outward currents on depolarization and the tail
current on repolarization (Fig. 1C). The current density of
the tail current after a depolarizing pulse to 20 mV in the ab-
sence and presence of 1, 10 and 30 mM NIP-142 was
9.7�0.7 pA/pF (n�10), 9.5�1.4 pA/pF (n�4), 5.4�1.0
pA/pF (n�5) and 1.8�0.5 pA/pF (n�5), respectively. The
EC50 value for the inhibition of tail current on repolarization
from 20 mV was 13.2 mM (Fig. 1E).
DISCUSSION
NIP-142 concentration-dependently blocked the
KCNQ1/KCNE1 channel current with an EC50 value of
13.2 mM (Fig. 1). The reported EC50 value of NIP-142 for the
GIRK1/4 channel current is 0.64 mM,9) that for the Kv1.5
channel current is 4.8 mM,10) and that for the HERG channel
current is 44 mM.9) The blocking potency of NIP-142 on the
KCNQ1/KCNE1 channel current was relatively low, suggest-
ing that the prolongation of atrial action potential by the
compound is mainly produced by blockade of potassium cur-
rents other than IKs, especially the acetylcholine-activated
potassium current. However, as the concentration range of
NIP-142 (10 to 100 mM) to prolong the atrial action poten-
tial8) overlaps that to inhibit the KCNQ1/KCNE1 channel
current (Fig. 1E), the possibility that IKs blockade contributes
to the effect of NIP-142 can not be totally excluded. The IKs
current is known to be increased by factors such as b-adren-
ergic stimulation12) or angiotensin II.13) A gain-of-function
mutation of KCNQ1 was reported to cause atrial fibrilla-
tion.14) Development of novel IKs blockers with antiarrhyth-
mic activity are in progress.15) Thus, the IKs-blocking effect
of NIP-142 may contribute to its antifibrillatory activity
under certain pathological conditions.
NIP-142 was reported not to prolong the action potential
duration in isolated guinea-pig ventricular tissue prepara-
tions,8) despite its inhibitory effect on the KCNQ1/KCNE1
channel current. One explanation for this apparent discrep-
ancy is the variation in sensitivity among experimental sys-
154 Vol. 34, No. 1
Fig. 1. Effect of Chromanol 293B and NIP-142 on KCNQ1/KCNE1
Channel Currents Expressed in HEK293 Cells
(A) Typical recordings in the absence (open circles) and presence of 1 mM (closed tri-
angles), 10 mM (closed squares) and 30 mM (closed circles) chromanol 293B. (B) Sum-
marized current–voltage relationships for the peak outward tail current on repolariza-
tion to �40 mV from a 3 s depolarization to various test potentials in the absence (open
circles) and presence of 1 mM (closed triangles), 3 mM (closed diamonds), 10 mM (closed
squares) and 30 mM (closed circles) chromanol 293B. (C) Typical recordings of the ef-
fects of NIP-142. (D) Summarized current–voltage relationships for the peak outward
tail current on repolarization to �40 mV from a 3 s depolarization to various test poten-
tials in the absence (open circles) and presence of 1 mM (closed triangles), 10 mM
(closed squares) and 30 mM (closed circles) NIP-142. (E) Concentration–response rela-
tionship for blockade by chromanol 293B (open circles) and NIP-142 (closed circles)
of the peak tail current on repolarization from 20 mV. Symbols and bars represent the
mean�S.E.M from 4 to 8 experiments.
tems. There are cases in which potency of ion channel in-
hibitors are higher in voltage-clamped single cells than in
myocardial tissue preparations (S(�)-Efonidipine16)). The ex-
tent of IKs-blockade by NIP-142 in tissue preparations might
be smaller than expected from the present results. Another
possible explanation for the lack of action potential prolon-
gation by NIP-142 in the ventricle is its blocking effect on
calcium channels. We have observed that NIP-142 has block-
ing effect on the L-type calcium channel,17) which may coun-
teract its action potential-prolonging effect through potas-
sium channel blockade. In any case, the details for the mech-
anisms of action of NIP-142 remain to be investigated.
Acknowledgments This study was supported in part by
the Science Research Promotion Fund from the Promotion
and Mutual Aid Corporation for Private Schools of Japan
(for Y. Tanaka and H. Tanaka). This study was partly per-
formed as a part of the project “Research on the molecular
mechanisms of appearance of age-related diseases by failure
of cell function control system, and their prevention and
treatment” by the “Research Center for Aging and Age-
Related Diseases” established in the Toho University Faculty
of Pharmaceutical Sciences.
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