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648 Acta Physiologica Sinica, October 25, 2005, 57 (5): 648-652
http://www.actaps.com.cn
Research Paper
Inhibitory effect of rhynchophylline on human ether-a-go-go related gene
channel
GUI Le 1 , LI Zhi-Wang 2 , DU Rong 3 , YUAN Guo-Hui 3 , LI Wei 3 , REN Fa-Xin 3 , LI Jing 3 , YANG Jun-Guo 3,*
1
Department of Cardiology, Affiliated Hospital, Nantong University, Nantong 226000, China; 2 Department of Neurobiology,
Huazhong University of Science and Technology, Wuhan 430030, China; 3 Institute of Cardiovascular Medicine, Union Hospital,
Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China
Abstract: We studied the effects of Chinese traditional medicine rhynchophylline (Rhy) on human ether-a-go-go related gene (HERG)
channel and characterized the electrophysiological properties of Rhy’s pharmacological effect on HERG channel using Xenopus
oocytes. Xenopus oocytes were injected with either 23 nl (5.75 ng) HERG cRNA or 23 nl distilled water. Xenopus oocytes were
randomly assigned to receive one of the following different concentrations of Rhy: (1) control, (2)10 µmol/L Rhy, (3)100 µmol/L Rhy,
(4) 500 µmol/L Rhy, (5) 1 000 µmol/L Rhy, (6) 10 000 µmol/L Rhy. Cell currents were recorded in oocytes. The peak tail currents of
HERG channel were inhibited by Rhy. The inhibition was in a dose-dependent manner [IC 50
=(773.4 ± 42.5) µmol/L]. Experiment with
100 µmol/L Rhy indicated that the degree of HERG blockade showed some voltage dependence (within –40 mV to –20 mV ). Kinetic
analyses revealed that Rhy decreased the rate of channel activation. The findings indicate that Rhy inhibits HERG encoded potassium
channels. It may underline the molecular mechanism of myocardial electrophysiological characteristics associated with this drug.
Key words: HERG; rhynchophylline; oocyte; two-microelectrode voltage clamp
human ether-a-go-go
1 2 3 3 3 3 3 3, *
1
226000 2 430030 3
430030
human ether-a-go-go (HERG)cRNA
(1) HERG IC 50
(773.4 ± 42.5) µmol/L(2)
HERG –20 mV 15% HERG
human ether-a-go-go
R966
Gouteng is a Chinese traditional medicine which belongs
to the plants of Rubinaceae genus. As a Chinese traditional
medicine, Gouteng was mainly used to treat ailments of
the cardiovascular and central nervous diseases, such as
hypertension, convulsions, numbness, and lightheadedness.
Rhynchophylline (Rhy) is the primary pharmacological
component of Gouteng [1] . The pharmacological
actions of Rhy on cardiovascular system were extensively
studied. It has been shown that Rhy has protective effect
on cardiac arrhythmias. Rhy can delay the atrio-ventricular
and the intraventricular conduction. The interval of P-
R, P-P, Q-T, and QRS waves are prolonged markedly [2,3] .
Some studies indicated that the electrophysiological effects
of Rhy result from the blockade of K + channel and L-type
Received 2004-09-06 Accepted 2005-07-27
*
Corresponding author. Tel: +86-27-85726810; Fax: +86-27-85726810; E-mail: ybemail@163.net

GUI Le et al: Inhibitory Effect of Rhy on HERG Channel
Ca 2+ channels [4,5] . However, the ionic mechanism of its
antiarrthymic action is not clear. I K1 , I to , I Ks and I Kr are major
components in the process of repolarization, so they
may be important targets for antiarrthymic drug action.
The effect of Rhy on I K1 , I to and I K has been reported, but
I Kr and I Ks has not been studied respectively. So we intend
to investigate the effects of Rhy on I Kr in this study.
Human ether-a-go-go related gene (KCNH2) encodes
the pore-forming subunit of the rapidly activating delayed
I Kr . I Kr is very important for the repolarization phase of the
cardiac action potential [6-8] . A wide range of other therapeutic
agents with diverse chemical structures can also
block the HERG channels [9-11] . So HERG channel is a significant
target for pharmacological management of
antiarrthymias. Understanding the molecular determinants
of the drug action on the HERG channels becomes meaningful
because of these unique pharmacological properties
of the HERG channel.
In order to clarify the pharmacological effect of Rhy on
K + channel, HERG channel (I Kr ) properties were studied
to determine whether Rhy is responsible for the dysfunction
of HERG channel (I Kr ). Heterologous expression system
of Xenopus oocyte was used to examine the channel
properties.
1 MATERIALS AND METHODS
649
1.1 In vitro transcription
The HERG cDNA subcloned into the BamH I-EcoR I site
of pGH19 vector was kindly provided by Dr. WANG Qing
(Cleveland Clinic Lerner College of Medicine of Case Western
Reserve University). HERG cDNA was linearized by
digestion with Not restriction enzyme, and cRNA was
prepared with In Vitro Transcription kit with T7 RNA polymerase
and Cap analog (Ambion, Austin, Tx, USA). cRNA
was dissolved in RNase free H 2 O. The cRNA concentration
was estimated by spectrophotometers (Thermo
Spectronic, USA) and diluted to desired concentration in
RNase free H 2 O before use.
1.2 Oocyte preparation and injection
The investigation conforms to the Guide for the Care and
Use of Laboratory Animals published by the Ministry of
Health of People’s Republic of China (No. 55, 1998). Female
Xenopus laevis frogs were anesthetized by ice for 30
to 60 min. Ovarian lobes were isolated and oocytes were
removed, washed in Ca 2+ -free ND96 solution (mmol/L:
NaCl 96, KCl 2, MgCl 2 1, HEPES 10, Na pyruvate 2.5, pH
7.5 adjusted with NaOH). Stage and Xenopus oocytes
were defolliculated by treatment with 2 mg/ml collagenase
(Sigma Aldrich Co.) for 30 to 60 min, and washed
with Ca 2+ -free ND96 solution. Oocytes were injected with
either 23 nl (5.75 ng) HERG cRNA or 23 nl distilled water
using automatic nanoliter injector (NANOI NJECT ,
Drummond Scientific Company). Injected oocytes were
incubated for 2~3 d at 18 °C in ND96 solution (mmol/L:
NaCl 96, KCl 2, CaCl 2 1.8, MgCl 2 1, HEPES 10, Na pyruvate
2.5, pH 7.5 adjusted with NaoH), supplemented with
penicillin (100 µg/ml) and streptomycin (100 µg/ml).
1.3 Rhy preparation
Rhy was dissolved in distilled water to give a 10 mmol/L
stock solution. Aliquots of stock solution were added to
ND96 solution to give the concentrations referred to in the
figures. The control was the solution of ND96 without
Rhy.
1.4 Electrophysiology recording and data analysis
Membrane currents in Xenopus oocytes were recorded by
two-microelectrode voltage clamp technique with amplifier
TURBO TEC-03X (NPI Electronic GmbH Company,
Germ any) at a temperature of 20~22 °C. Oocytes were
individually assigned into different concentrations of Rhy.
Current injecting electrode and potential measuring electrode
were filled with 3 mol/L KCl. The resistances of these
electrodes were 0.5~2.0 MΩ. The bath solution was connected
to the ground electrically through a low-resistance
agar bridge. Pulse protocols are described in the figure
legends. Only oocytes whose resting potential were around
–40 mV were used. Cell work 5.0, cell work reader 5.0
softwares and Sigmaplot 8.0, Clampfit 8.0 softwares were
used for data acquisition and data analysis respectively.
The data of concentration dependence was analyzed using
the equation: Fraction inhibition = 1– (I Rhy /I control ), and fitted
with the Hill equation: Fraction inhibition = 1/(1+ (IC 50 /
[Rhy]) h ), where I Rhy is the tail currents at – 60 mV after a
depolarization pulse to + 20 mV recorded in different concentrations
(10, 100, 500, 1 000 and 10 000 µmol/L ),
I control is the tail current at –60 mV after a depolarization
pulse to +20 mV recorded in control, [Rhy] is Rhy
concentration, IC 50 is [Rhy] producing half maximal inhibition
of tail currents, h is the Hill coefficient.
The voltage dependence of HERG current activation was
determined for each oocyte by fitting tail current amplitudes
(I tail ) versus test potential to a Boltzmann function in
the following equation: I tail = I tail-max /{1+exp[(V 1/2 -V t )/k]},
where I tail-max is peak I tail , V t is test potential, V 1/2 is the
voltage at which I tail is half of I tail-max , and k is slope factor.
1.5 Statistical analysis

650
All values are expressed as means ± SEM. Differences
within these values were evaluated by ANOVA and t-test.
P

GUI Le et al: Inhibitory Effect of Rhy on HERG Channel
651
Fig.3. Voltage dependence of Rhy suppression effect. A: I-V relationship
for amplitudes of tail currents recorded during depolarizing
pulses in control (closed circle) and 100 µmol/L Rhy (open circle)
groups. B: Normalized I-V relationships for mean amplitudes of tail
currents in control (closed circle) and in 100 µmol/L Rhy (open
circle) groups. C: Mean level of fractional block [1– (I Rhy /I control )] was
plotted against test pulse voltage.
Rhy group (1 000 µmol/L), the inhibition level of tail current
by Rhy (10 µmol/L) , Rhy (100 µmol/L) and Rhy
(1 000 µmol/L) were (9.5 ± 7.48)%, (16.2 ± 5.87)% and
(72.62 ± 2.30)% (means ±SEM, P

652
about 15% at –20 mV. These findings suggest that Rhy
binds to HERG channels that are in the open state.
This is the first time that the sensitivity of the HERG
channel to Rhy has been demonstrated. It is reasonable to
conclude that action potential prolongation with Rhy is due
to the inhibitory action of Rhy on the HERG channel. Rhy
inhibits HERG channels in a voltage-dependent as well as
dose-dependent manner. The potent inhibition of HERG
channels by Rhy underlies its potential effectiveness against
ventricular tachyarrhythmias in clinical practices.
* * *
ACKNOWLEDGEMENTS: We thank Dr. WANG Qing
(The Cleveland Clinic Foundation) for the HERG cDNA.
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* * * * * *
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