Clinical Science (2004) 107, 175–182 (Printed in Great Britain)
Probucol aggravates long QT syndrome
associated with a novel missense mutation
M124T in the N-terminus of HERG
Kenshi HAYASHI∗ , Masami SHIMIZU∗ , Hidekazu INO∗ , Masato YAMAGUCHI∗ ,
Hidenobu TERAI∗ , Naoto HOSHI†, Haruhiro HIGASHIDA†, Nariaki TERASHIMA‡,
Yoshihide UNO‡, Honin KANAYA‡ and Hiroshi MABUCHI∗
∗
Molecular Genetics of Cardiovascular Disorders, Division of Cardiovascular Medicine, Graduate School of Medical Science,
Kanazawa University, Kanazawa, Japan, †Biophysical Genetics, Division of Neuroscience, Graduate School of Medical
Science, Kanazawa University, Kanazawa, Japan, and ‡Division of Cardiology, Ishikawa Prefectural Hospital, Kanazawa, Japan
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Patients with LQTS (long QT syndrome) with a mutation in a cardiac ion channel gene, leading
to mild-to-moderate channel dysfunction, may manifest marked QT prolongation or torsade de
pointes only upon an additional stressor. A 59-year-old woman had marked QT prolongation
and repeated torsade de pointes 3 months after initiation of probucol, a cholesterol-lowering
drug. We identified a single base substitution in the HERG gene by genetic analysis. This novel
missense mutation is predicted to cause an amino acid substitution of Met124 → Thr (M124T) in the
N-terminus. Three other relatives with this mutation also had QT prolongation and one of them
had a prolonged QT interval and torsade de pointes accompanied by syncope after taking probucol.
We expressed wild-type HERG and HERG with M124T in Xenopus oocytes and characterized
the electrophysiological properties of these HERG channels and the action of probucol on the
channels. Injection of the M124T mutant cRNA into Xenopus oocytes resulted in expression of
functional channels with markedly smaller amplitude. In both HERG channels, probucol decreased
the amplitude of the HERG tail current, decelerated the rate of channel activation, accelerated the rate of channel deactivation and shifted the reversal potential to a more positive
value. The electrophysiological study indicated that QT lengthening and cardiac arrhythmia in the
two present patients were due to inhibition of IKr (rapidly activating delayed rectifier K+ current)
by probucol, in addition to the significant suppression of HERG current in HERG channels with
the M124T mutation.
INTRODUCTION
Congenital LQTS (long QT syndrome) is characterized
by prolonged ventricular repolarization and a propensity
for life-threatening ventricular tachyarrhythmias, resulting in syncopal attacks and sudden death [1]. Mutations
in various cardiac ion channel genes, including KCNQ1,
KCNE1, HERG, KCNE2 and SCN5A [2–8], are known
to cause this syndrome, and many LQTS mutations have
been identified [9]. HERG encodes a voltage-gated K+
channel and the HERG current is similar to IKr (rapidly
activating delayed rectifier K+ current) [10] observed in
the myocardium. Cellular expression studies revealed that
mutations in different regions of the HERG gene elicit
a variety of functional changes in the HERG channel
[11], suggesting that each mutation should be examined
Key words: arrhythmia, genetics, ion channel, long QT syndrome, probucol, torsade de pointes.
Abbreviations: Erev , reversal potential; IKr , rapidly activating delayed rectifier K+ current; I–V relationship, current–voltage
relationship; LQTS, long QT syndrome; QTc, QT interval corrected for heart rate; SSCP, single-strand conformational
polymorphism; V 1/2 , voltage at which the current was half-activated; WT, wild-type.
Correspondence: Dr Kenshi Hayashi (email [email protected]).
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to clarify the mechanism of suppression of the HERG
current.
In patients with LQTS caused by a mutation, leading to
mild-to-moderate dysfunction of a cardiac ion channel,
marked QT prolongation or torsade de pointes may appear only upon the addition of a stressor, such as a drug,
hypokalaemia or bradycardia. Probucol, a cholesterollowering drug, is known to induce QT prolongation in
some patients, although ventricular arrhythmia or syncope due to the drug is rare [12].
We have recently identified in a Japanese family with
LQTS a novel missense mutation in the N-terminus of
the HERG gene that resulted in a putative amino acid
substitution of Met124 → Thr (M124T). While taking
probucol, two family members with this mutation had
QT prolongation and torsade de pointes with recurrent
syncope. Since this particular mutation of the HERG
gene has not been reported previously and since the relationship between QT prolongation and the action of
probucol has not been clarified, it is of interest to identify whether this mutation causes HERG channel dysfunction and how probucol prolongs the QT interval.
Using the heterologous expression system of Xenopus
oocytes, we examined the properties of this mutant channel and the effect of probucol on both WT (wild-type)
and mutant M124T HERG channels. We demonstrate
that the M124T mutation in HERG causes IKr dysfunction, and that probucol decreases the current amplitudes
of both WT and mutant HERG channels by altering the
activation and deactivation kinetics and ion selectivity.
METHODS
DNA isolation and mutation analysis
Genetic analysis was performed after obtaining informed
consent from all subjects. Genomic DNA was purified
from the white blood cells of seven family members,
after which PCR amplification was performed [13]. SSCP
(single-strand conformational polymorphism) analysis of
the amplified DNA was performed to screen for mutations in KCNQ1, HERG, SCN5A, KCNE1 and KCNE2
as described previously [14] with a slight modification
[15]. Normal and aberrant SSCP products were isolated
and sequenced by ABI PRISM 310 (PerkinElmer Applied
Biosystems, Foster City, CA, U.S.A.). We sequenced all
15 exons of the HERG gene in the proband using 19 primer pairs as described previously [16]. To confirm the
missense mutation that served as the basis of the present
study, restriction enzyme analysis was performed. Using
a mutagenic primer, on PCR gene amplification we
introduced an artificial AccII site in the PCR product
of the only T → C allele (M124T). Digestion of the PCR
products derived from the mutant allele with AccII gave
rise to a 119 bp fragment instead of a 138 bp fragment
when resolved on a polyacrylamide gel.
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Site-directed mutagenesis and in vitro
transcription of cRNA
HERG cDNA in a pGH19 vector was kindly provided by
Dr Gail A. Robertson (Department of Physiology, University of Wisconsin, Madison, WI, U.S.A.). The M124T
HERG cDNA was constructed by an overlap extension strategy [17]. The sequence of the M124T HERG
cDNA was confirmed by DNA sequence analyses
using 11 primer pairs. WT HERG cDNA and M124T
HERG cDNA were linearized by digestion with Not I,
and cRNAs were prepared with the mMESSAGE
mMACHINE kit (Ambion, Austin, TX, U.S.A.) using
T7 RNA polymerase.
Electrophysiology
Defolliculated Xenopus lavies oocytes (stage V–VI) were
isolated as described previously [18]. Each oocyte was
injected with 50 nl of solution containing 1.0 ng of
WT HERG cRNA, 1.0 ng of M124T HERG cRNA
or a combination of 1.0 ng of WT HERG cRNA and
1.0 ng of M124T HERG cRNA. The oocytes were
incubated at 16 ◦ C in modified Barth’s solution (87.4 mM
NaCl, 1 mM KCl, 2.4 mM NaHCO3 , 10 mM Hepes,
0.82 mM MgSO4 , 0.66 mM NaNO3 and 0.74 mM CaCl2 ,
pH 7.5) supplemented with penicillin (100 µg/ml) and
streptomycin (100 µg/ml). The oocytes were used for
experiments 2–4 days after injection.
Membrane currents were studied using the twomicroelectrode voltage clamp technique with an amplifier
(AXOCLAMP-2A; Axon Instruments, Foster City,
CA, U.S.A.) at room temperature of 23–25 ◦ C as described previously [19]. During recording, oocytes were
perfused continuously with ND96 solution (96 mM
NaCl, 2 mM KCl, 5 mM MgCl2 , 0.3 mM CaCl2 and
5 mM Hepes, pH 7.6). Probucol (kindly supplied by
Daiichi Pharmaceutical, Tokyo, Japan) was stored at 4 ◦ C.
On the day of the experiment, probucol was dissolved in
ND96 solution and the pH was re-adjusted to 7.6. All
measurements were made under steady-state conditions
at least 3 min after total solution exchange.
Data acquisition and analysis were performed by a
Digi DATA 1200 A/D converter (Axon Instruments) and
pCLAMP (version 5.5.1; Axon Instruments).
Voltage clamp protocols and
data analysis
All pulse protocols are described in the Figure legends.
Data analysis was carried out using Clampfit (version 6.1;
Axon Instruments). The voltage-dependence of HERG
current activation was determined for each oocyte by
fitting the peak value of Itail (tail current) against the
test potential to a Boltzmann function in the following
formula:
Itail = Itail-max / 1 + exp V1/2 − Vt /k
Probucol aggravates long QT syndrome
Figure 1 ECGs of the proband (A) and the proband’s niece (B) before and after initiation of probucol
(A) Control ECGs obtained before probucol was prescribed show a prolonged QTc (495 ms). ECGs obtained approx. 2 months after initiation of probucol show a
markedly prolonged QTc (550 ms) and torsade de pointes. (B) Control ECGs obtained before probucol was prescribed show a prolonged QTc (478 ms). ECGs obtained
approx. 3 months after initiation of probucol show marked QTc prolongation (660 ms) and torsade de pointes.
where Itail-max is the peak Itail , V t is the test potential, V 1/2
is voltage at which the current was half-activated (i.e. at
which Itail is half of Itail−max ), and k is the slope factor.
All values are expressed as means +
− S.E.M. Differences
among these values were evaluated by ANOVA and the
unpaired Student’s t-test where appropriate. P < 0.05 was
considered to be significant.
RESULTS
Clinical diagnosis
The proband was a 59-year-old woman who experienced
recurrent syncope. She had started taking probucol
(500 mg daily) for hypercholesterolaemia approx.
2 months prior to the episode of syncope. Figure 1(A,
upper left panel) shows the ECG of the proband (Figure 2B, II-1) obtained approx. 8 years prior to initiation
of probucol, which exhibited a prolonged QT interval.
QTc (QT interval corrected for heart rate using Bazett’s
formula = QT/square root of RR interval in seconds)
was 495 ms. On the ECG obtained at admission, she
had a significantly prolonged QTc interval of 550 ms
(Figure 1A, upper-right panel) and polymorphic ventricular tachycardia (torsade de pointes; Figure 1A,
lower panel), accompanied by fainting. The proband
was diagnosed with LQTS. Probucol was discontinued
soon after admission; however, the QTc interval remained
prolonged (646 ms) 50 days after discontinuation of
probucol. Figure 1(B, upper-left panel) shows the ECG
of a niece of the proband (Figure 2B, III-4), which had
been obtained when she was not taking any medication,
indicating a prolonged QTc interval (478 ms). She was
a 19-year-old woman who had been diagnosed with
xanthomatosis and was prescribed 500 mg of probucol
daily. Three months after she started taking probucol, she
experienced an episode of syncope. An ECG revealed a
markedly prolonged QTc interval (660 ms; Figure 1B,
upper-right panel) and torsade de pointes (Figure 1B, lower panel). The concentration of probucol in
her blood at the time of having cardiac arrhythmia was
26 µg/ml, which was within the therapeutic range. The
two sisters (Figure 2B, II-2 and II-4) of the proband
had prolonged QTc intervals of 524 ms and 478 ms
respectively, on ECGs obtained when they were not
taking any drugs. The average QTc interval of these four
patients before taking probucol was 494 +
− 11 ms.
Molecular genetic analyses
Screening for mutations in the HERG gene by SSCP
analysis identified an abnormal SSCP conformer in the
DNA samples from the proband. This SSCP anomaly
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were expressed in oocytes and the resultant currents were
analysed. Moreover, we investigated the interaction of
probucol with WT and mutant HERG channels.
Electrophysiological characterization
of the M124T HERG mutant
Figure 2 Mutation analysis of the HERG gene and pedigree
of the proband
(A) DNA sequence analysis of the HERG gene in the proband. A single nucleotide
transition from T → C at nucleotide position 371 in the HERG gene was found
in the proband and in three relatives. (B) The pedigree and QTc intervals.
The arrow in the pedigree indicates the proband. Numbers indicate the length
of the QTc interval (ms). 䊉, heterozygous female patients with the HERG
M124T mutation. (C) PCR-RFLP analysis. Digestion of the PCR products with Acc II
generated polymorphic restriction fragments of 138 bp and/or 119 bp.
was not observed in DNA samples from more than
100 control subjects. Sequence analysis of the abnormal
SSCP conformer revealed a single base substitution (T →
C) at nucleotide position 371 (Figure 2A). On PCR
RFLP (restriction fragment length polymorphism) analysis, all four individuals with prolonged QTc intervals showed both 138 bp and 119 bp fragments, indicating heterozygosity for the mutation (Figure 2C).
This mutation is predicted to result in an amino acid
substitution of Met124 → Thr (M124T), which is located
in the N-terminus of the HERG subunit. Sequence
analysis of all 15 exons of the HERG gene did not
show any other mutations. Screening for mutations in
the KCNQ1, SCN5A, KCNE1 and KCNE2 genes by
SSCP did not show any abnormal SSCP conformers. In
order to obtain information regarding how and which
dysfunction is elicited by this mutation, we constructed M124T mutant HERG cDNA. We confirmed that the
M124T HERG cDNA contained no other mutation by
DNA sequence analysis. WT and mutant HERG cRNAs
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Oocytes injected with 1.0 ng of WT HERG cRNA alone
showed a slowly activating outward current that was
associated with an inwardly rectifying property (Figures 3A and 3E), as described previously [10,20]. In
contrast, oocytes injected with 1.0 ng of M124T HERG
cRNA alone showed these currents, although the currents
were smaller than those in oocytes injected with WT
HERG cRNA alone (Figures 3B, 3E and 3F). The mean
amplitude of the tail currents measured at − 60 mV after
a depolarizing test pulse to + 20 mV was 521 +
− 105 nA
(n = 8) for the M124T HERG channels, which was
significantly smaller than that of 1839 +
− 216 nA (n = 10)
for the WT HERG channels (P < 0.01). When M124T
and WT HERG channels were co-expressed by injecting
equal amounts of WT and M124T HERG cRNA into
oocytes (i.e. 2.0 ng of cRNA in total; M124T/WT),
the evoked currents were nearly the same as those in
oocytes injected with WT HERG cRNA alone. The mean
amplitude of the tail currents measured at − 60 mV after
a depolarizing test pulse to + 20 mV was 2223 +
− 161 nA
(n = 16) for the WT channels and 2377 +
159
nA
(n = 14)
−
for the M124T/WT channels (results not shown). Thus
the M124T HERG channel did not suppress the WT
HERG channel in a dominant-negative manner.
The amplitude of the tail currents was plotted as a
function of the test potential and the curve was fitted
to a Boltzmann function (Figure 4A). V 1/2 was − 17.6 +
−
1.4 mV (n = 7) for the WT channels and − 20.3 +
− 1.2 mV
(n = 7) for the M124T channels. The slope factor was
8.4 +
− 0.6 mV (n = 7) for the WT channels and 7.8 +
−
0.2 mV (n = 7) for the M124T channels. V 1/2 and slope
factor were comparable between the WT and M124T
channels (P = 0.20 and P = 0.34 respectively). These data
suggest that this mutation does not affect the voltagedependence activation of the HERG channel.
Deactivation of currents following test pulses could
be fitted to a double exponential function. The fast (at
test potentials between − 80 and − 30 mV) and slow
(between − 60 and − 20 mV) time constants of deactivation of the M124T HERG channels were similar to those
of the WT HERG channels (Figures 4B and 4C). Our
results are consistent with those of a previous report [21]
in which some mutations close to the transmembrane in
the N-terminus region of the HERG gene had little or no
effect on the time course of deactivation of the expressed
HERG channel.
Erev (reversal potential) was determined by clamping
the tail currents at potentials ranging from − 120 mV
to + 10 mV in 10 mV increments after a 3 s test pulse to
Probucol aggravates long QT syndrome
Figure 3 Voltage-clamp recordings of Xenopus oocytes expressing WT or M124T HERG (A–D) and I –V relationship of the
peak currents (E,F) without and with probucol
(A–D) Voltage-clamp recordings of Xenopus oocytes expressing WT or M124T HERG before and after bath superfusion with probucol. Representative whole-cell current
traces of oocytes that had been injected with 1.0 ng of WT cRNA (A) or 1.0 ng of M124T cRNA (B) in the absence of probucol, or 1.0 ng of WT cRNA (C) or 1.0 ng
of M124T cRNA (D) in the presence of 30 µM probucol, are shown. Depolarizing pulses were applied to a potential ranging between − 80 and + 50 mV for 3 s,
followed by a hyperpolarizing pulse to − 60 mV for 6 s. Holding potential of − 60 mV was used. To show the current traces clearly, selected traces elicited by five
depolarizing pulses (− 60, − 40, − 20, + 10 and + 40 mV) are displayed. (E) I –V relationship of the peak currents during the test pulse. (F) I –V relationship of
the amplitudes of the peak tail currents. (E and F) WT (䊏) and M124T (䊉) before bath superfusion with probucol; WT (䊐) and M124T (䊊) after bath superfusion
with probucol. At each test potential, eight or ten different oocytes were used.
+ 20 mV. Erev of M124T HERG channels was − 86.9 +
−
1.2 mV (n = 13), which was similar to that of WT channels
(− 87.4 +
− 2.8 mV; n = 12).
Effects of probucol on WT and M124T
HERG channels
We next investigated the effect of probucol on both the
WT and M124T HERG channels. Current amplitudes
and the activation and deactivation kinetics of HERG
currents were analysed in both the absence and presence
of 30 µM probucol. This concentration of probucol cor-
responded to the serum concentration of probucol at
8–12 weeks of continuous treatment with probucol
(1 tablet of 500 mg twice daily) in patients with familial
hypercholesterolaemia [22].
Figure 3(C and D) shows the original currents obtained after 3 s depolarizing pulses from a holding
potential of − 80 mV up to + 50 mV in 10 mV steps
in the presence of 30 µM probucol. In oocytes injected
with either WT or M124T HERG cRNA, probucol
did not affect the peak current amplitudes, but it
shifted the voltage at the peak amplitude to a positive
potential by approx. 10 mV in both the WT and M124T
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158 nA for WT to 967 +
− 146 nA for WT + probucol;
n = 10 each; P < 0.01) and by 47.2 +
− 9.3 % through
the M124T channels (from 509 +
97
− nA for M124T
to 260 +
81
nA
for
M124T
+
probucol;
n = 8 each,
−
P < 0.05; Figure 3F). Probucol blocked the WT and
M124T channels by a similar degree (P = 0.44).
Normalized tail currents through the WT and
M124T channels in the control and 30 µM probucoltreated conditions are shown in Figure 4(A). Perfusion of oocytes with probucol significantly shifted V 1/2
of activation of WT channels from − 17.6 +
− 1.4 mV to
− 8.2 +
1.7
mV
(n
=
7;
P
<
0.01).
Similarly,
upon treat−
ment with probucol, V 1/2 of activation of M124T HERG
channels significantly shifted from − 20.3 +
− 1.2 mV to
− 9.5 +
2.4
mV
(n
=
7;
P
<
0.01).
Thus
probucol
decel−
erated the rate of HERG channel activation.
Probucol accelerated the deactivation rate of both the
WT and M124T HERG channels (Figures 3C, 3D, 4B and
4C). Application of 30 µM probucol to both channels
significantly decreased the fast time constants at test
potentials between − 70 and − 40 mV and the slow time
constants at test potentials between − 60 and − 30 mV.
Thus probucol accelerated the deactivation of HERG
channels.
Application of 30 µM probucol significantly shifted
the Erev of WT HERG channels from − 87.4 +
− 2.8 mV
(n = 12) to − 77.6 +
2.4
mV
(n
=
8;
P
<
0.05).
Similarly,
−
30 µM probucol significantly shifted Erev of M124T
HERG channels from − 86.9 +
− 1.2 mV (n = 13) to
− 74.2 +
− 2.1 mV (n = 9; P < 0.01). These results suggest
that probucol alters the selectivity for K+ .
Figure 4 Normalized I –V relationships of the mean amplitudes of the peak tail currents (A) and deactivation time
constants of HERG currents (B,C)
(A) Normalized I –V relationships of the mean amplitudes of the peak tail currents.
At each test potential, 8–11 different oocytes were used. Curves represent best
fits to a Boltzmann function. (B and C) Deactivation time constants of HERG
currents. To examine the time course of deactivation, a current was activated
with a 3 s pulse to + 20 mV, followed by a return to the test potential ranging
from − 80 mV to − 20 mV (in 10 mV steps; inset). The deactivation process
was fit to bi-exponential functions. Plots of the fast (B) and slow (C) components
of the deactivation time constant against test potential are shown. The values are
∗
the means +
− S.E.M. of 13–20 different oocytes. P < 0.05 compared with WT
or M124T in the absence of probucol. WT (䊏) and M124T (䊉) in the absence
of probucol; WT (䊐) and M124T (䊊) in the presence of probucol.
channels (Figure 3E). This positive shift suggested that
probucol affected the voltage-dependence of HERG
channel activation. In contrast, probucol significantly
decreased the peak tail current amplitude measured at
− 60 mV after a depolarizing test pulse to 0 mV by
38.3 +
− 6.4 % through the WT channels (from 1581 +
−
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DISCUSSION
We identified in a Japanese family with LQTS a novel
single base substitution (from T → C) at nucleotide position 371 in the HERG gene. This mutation is assumed to
lead to the substitution of Met124 → Thr, which is located
in the N-terminus of HERG channels (Figure 2A).
Because the M124T mutant was able to produce
HERG outward currents when the corresponding cRNA
was injected to express M124T HERG in Xenopus
oocytes (Figure 3B), homomultimers of M124T subunits
appeared to form functional channels. The amplitude of
the outward tail current through M124T channels was less
than one-third of that through the WT HERG channels.
Moreover, upon co-expression of M124T with WT in
oocytes, this mutant subunit did not have a dominantnegative effect on the WT subunit. Many properties of
the WT and M124T HERG currents, including the peak
of the current–voltage (I–V) relationship for activating
currents recorded during depolarizing pulses (Figure 3E), the voltage dependence of activation (Figure 4A)
and the time course of deactivation (Figures 4B and 4C),
were similar. In previous reports [21,23], some mutations
Probucol aggravates long QT syndrome
occurring around the PAS (Per-Arnt-Sim) domain in
HERG accelerated HERG channel deactivation. For
example, the rate of deactivation was significantly faster
in HERG mutants with deletion of nucleotide residues
2–23 or 2–26 in HERG. However, mutations that were
located near codon 124 barely altered HERG channel
deactivation [21].
In the present study, all four patients with M124T
mutation in HERG had prolonged QTc intervals
(494 +
− 11 ms), but were clinically asymptomatic prior
to probucol administration; symptoms appeared in the
proband and her niece after they received probucol.
These clinical findings are consistent with the results
in the electrophysiological study in that the M124T
channels did not strongly suppress the WT channels
in a dominant-negative manner. A previous report [24]
found that LQTS patients with mutations in the non-pore
regions of HERG had a lower frequency of arrhythmiarelated cardiac events occurring at an earlier age than
those with mutations in the pore region. In some
individuals, congenital LQTS associated with a LQTSrelated ion channel gene mutation, leading to mild-tomoderate channel dysfunction, is not detected until they
are exposed to an additional stressor, such as particular
drugs, hypokalaemia or bradycardia [8,25–28].
The proband and her niece had a significantly prolonged QTc interval and torsade de pointes several
months after initiation of probucol. Probucol is known to
induce QT prolongation in some patients. In an analysis
of 89 adult patients treated for hypercholesterolaemia
with probucol, 23 patients (26 %) had QTc interval
prolongation (over 0.450 s) on ECG [12]. However,
the mechanism through which probucol prolongs the
QTc interval had not been clarified. The present study
showed that probucol blocks the HERG K+ channel.
Probucol inhibited both the WT and mutant HERG
tail currents (Figures 3C, 3D and 3F), induced a
positive shift of the activation curve (Figure 4A), accelerated deactivation (Figures 4B and 4C) and shifted
reversal potential to a more positive value, which can
decrease the net outward current during repolarization.
In the proband and her niece, the M124T mutation in
HERG caused IKr dysfunction and probucol further
suppressed the IKr current; this would cause a delay
in the repolarization phase of action potentials along
cardiomyocytes and induce QT prolongation and cardiac
arrhythmia. Moreover, the results of our electrophysiological study showed that probucol also inhibited
WT HERG channels, suggesting that this drug might
induce acquired LQTS by blocking the IKr . These
drug actions are similar to those of propoxyphene,
a relatively weak µ opioid receptor agonist, and its
metabolite, norpropoxyphene. Ulens et al. [29] reported
that propoxyphene and norpropoxyphene altered gating
of HERG channels by decreasing the channel activation
rate constant, increasing the channel deactivation rate
constant and shifting the reversal potential of HERG
currents to a more positive value. They also suggested that
the interaction of propoxyphene and norpropoxyphene
with amino acid Ser631 of the HERG channel protein
might contribute to the drug-induced alteration of ion
selectivity. We therefore postulate that probucol may act
on HERG channels in the same manner as propoxyphene
and norpropoxyphene. Although HERG tail currents
were decreased by probucol, the peak currents during the
pulse were not. HERG channels possess the feature that
rapid voltage-dependent inactivation is present during
the pulse, but relieved during repolarization, giving rise
to the resurgent tail current. The shift in inactivation
kinetics may result in the difference between the two
sets of measurements. It should be noted that the
potency of drug inhibition of the HERG current obtained
using an oocyte expression system may considerably
underestimate the actual drug potency towards the
HERG current in a mammalian expression system/native
tissue [30]. Therefore the level of inhibition of HERG
current by probucol obtained in the present study may
be lower than its actual level.
The proband in the present study had a prolonged
QTc interval (646 ms) 50 days after discontinuation of
probucol. Probucol remains in the body for a long period
of time. In a previous case report [31], probucol was still
detected in the plasma of the patient 3 months after its
discontinuation. The serum concentration of probucol
in the proband in the present study was not measured;
however, probucol may have still been present in her
body and prolonged the QT interval at 50 days after its
withdrawal.
In conclusion, we have identified a novel missense
mutation in the N-terminus of HERG in a Japanese
family with LQTS in which two family members developed torsade de pointes after taking probucol. The
results of our electrophysiological studies indicate that
QT prolongation and cardiac arrhythmia in the two
patients were due to IKr dysfunction as a result of
the M124T mutation in HERG and to the additional
inhibitory effect of probucol on cardiac IKr current.
Probucol also inhibited WT HERG channels and might
induce acquired LQTS.
Study limitation
Probucol is a lipid-soluble drug and has low solubility
in water. In the present study, we dissolved probucol in
ND96 solution mechanically using an ultrasonic homogenizer, followed by heat treatment at over 120 ◦ C.
Although these solution treatments could almost
completely dissolve the drug in water, the concentration
of the drug in the solutions was not measured. However,
it is hypothesized that this would not have a large effect
on our results, because the conditions were similar for the
WT and mutant HERG channels.
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ACKNOWLEDGMENT
We thank Dr Gail A. Robertson at the University of
Wisconsin for providing us with the HERG cDNA.
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Received 27 October 2003/22 March 2004; accepted 25 March 2004
Published as Immediate Publication 25 March 2004, DOI 10.1042/CS20030351
C
2004 The Biochemical Society