Cardiovascular Research 51 (2001) 670–680
www.elsevier.com / locate / cardiores
A spectrum of functional effects for disease causing mutations in the
Jervell and Lange-Nielsen syndrome
a
b
c
a,
Lingqian Huang , Maria Bitner-Glindzicz , Lisbeth Tranebjærg , Andrew Tinker *
a
Centre for Clinical Pharmacology, Department of Medicine, University College London, The Rayne Institute, 5 University Street,
London WC1 E 6 JJ, UK
b
Clinical and Molecular Genetics Unit, Institute of Child Health, 30 Guildford Street, London WC1 N 1 EH,
and Great Ormond Street Hospital for Children NHS Trust, Great Ormond Street, London WC1 N 3 JH, UK
c
Department of Medical Genetics, University Hospital Tromsø, Tromsø, Norway
Received 24 January 2001; accepted 11 May 2001
Abstract
Objective: Jervell and Lange-Nielsen syndrome (JLNS) is a recessively inherited long QT syndrome (LQTS) characterised by
profound sensorineural deafness and predisposition to syncope and sudden cardiac death. Mutation analysis has established the presence
of mutations in affected individuals in the genes KCNQ1 and KCNE1: the potassium channel complex responsible for the cardiac I Ks
current involved in repolarisation of the ventricular action potential. Our objective was to determine the functional effects of disease
causing mutations in JLNS. Methods: In this study we have investigated the electrophysiological effects of eight distinct JLNS mutations
after expression of cRNA in Xenopus laevis oocytes. Results: KCNE1 mutant T59P/ L60P showed no dominant negative effect and was a
pure loss of function mutation. KCNQ1 mutant E261D showed a strong dominant-negative effect. KCNQ1 mutant R243H produced a
moderate dominant-negative effect, right shifted the steady-state activation curve and led to an increased deactivation rate. The behaviour
of KCNQ1 mutants 572–576del, 1008delC, R518X, Q530X, R594Q depended on the relative quantities of mutant and wild-type proteins
(with a weak dominant-negative effect present at 1:3 but not 1:1 injection ratios). These data indicate the presence of an additional
assembly domain before S2–S3 and the importance of the S4–S5 region in channel function and gating. Conclusions: Our data suggest a
spectrum of behaviour for disease causing mutations from simple loss of function through to prominent dominant negative behaviour.
 2001 Elsevier Science B.V. All rights reserved.
Keywords: Arrythmia (mechanisms); Long QT syndrome; K-channel; Repolarization; Sudden death
This article is referred to in the Editorial by A.A.M.
Wilde and D. Escande ( pages 627 – 629) in this issue.
1. Introduction
Long QT syndrome (LQTS) is characterised by
prolongation of the QTc interval (QT interval corrected for
heart rate) on the electrocardiogram. It predisposes to
syncope, seizures and sudden death due to the development of a characteristic ventricular tachycardia known as
*Corresponding author. Tel.: 144-20-7679-6174; fax: 144-20-76796212.
E-mail address: [email protected] (A. Tinker).
torsades de pointes and subsequently fatal ventricular
fibrillation. Long QT syndromes can be acquired in origin
most commonly due to drugs or more rarely occur as a part
of an inherited syndrome. The autosomal recessive Jervell
and Lange-Nielsen syndrome (JLNS) was first described in
1957 and is characterised by profound sensorineural
deafness in association with a prolonged QT interval [1].
The more common Romano Ward syndrome (RWS) is
inherited in autosomal dominant fashion and patients have
only cardiac symptomatology with normal hearing [2,3].
Advances in molecular genetics have established mutations in genes encoding the cardiac sodium channel and
two potassium channels and their ancillary proteins [4–9].
Time for primary review 28 days.
0008-6363 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved.
PII: S0008-6363( 01 )00350-9
L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
JLNS and RWS are allelic at the molecular level as
homozygous mutations in the potassium channel gene
KCNQ1 cause JLNS [9–12] and heterozygous mutations
in KCNQ1 cause RWS [11,13–19]. Mutations in the gene
encoding a regulatory protein KCNE1 are also known to
cause both JLNS and RWS [7,9,20,21]. KCNQ1 together
with KCNE1 encodes the slowly activating delayed rectifier potassium current (I Ks ) channel; a current responsible
for repolarisation of the cardiac action potential [22,23].
Clinical and genetic data suggest that the functional
effects of mutations ascertained through RWS families
may differ from those ascertained through JLNS families.
Clinically, carriers of JLNS mutations are only occasionally symptomatic, although they may have prolonged QT
intervals [24]. Consequently the mild phenotypic effects of
these mutations may allow them to breed to homozygosity
in the population. Of the mutations causing RWS reported
to date on the LQTS databases (HUGO mutation database
initiative, Long QT syndrome database (LQTS database):
http: / / www.ssi.dk / en / forskning / lqtsdb / kvlqt1.htm; The
European Society of Cardiology database: http: / /
pc4.fsm.it:81 / cardmoc), the majority are predicted to have
a localised effect on the protein. These are mainly missense, with some in-frame deletions and splice mutations
(detailed analysis of which predicts translation of an inframe protein [25]), and only a few cause frameshift. As
RWS is a more common condition than JLNS, there has
been more opportunity to study the functional effects of
mutations associated with RWS, which have been shown
to act in a strong dominant negative manner. So far, only
three JLNS mutations (R243H, W305S, D544) have been
studied functionally [11,15,18,26]. There is no clear consensus on how these mutations cause their functional
effects. In this study, we report the functional effects of
eight JLNS mutations.
2. Methods
2.1. Site-directed mutagenesis and cRNA synthesis
Two human KCNQ1 cDNA clones were used in the
present study: short KCNQ1 (s-KCNQ1) [23] and long
KCNQ1 (l-KCNQ1) [15]. Human synthetic KCNE1 clone
was obtained from Dr Richard Swanson. Vectors pSP64,
pGEM3ZHE and pGEMA were used for the expression of
s-KCNQ1, l-KCNQ1 and KCNE1 cDNA clones, respectively. In this study we largely examined s-KCNQ1 as this
expressed robust currents in Xenopus laevis oocytes after
cRNA injection. However reasonable current expression
from the l-KCNQ1 cDNA was achieved after synthesis and
injection of cRNA generated after subcloning into the
pGEMHE vector [27]. The pGEMHE vector contains the
59 and 39 untranslated regions from the Xenopus laevis
beta globin gene and this can significantly increase currents. KCNQ1 and KCNE1 mutations were introduced into
671
wild type (WT) s-KCNQ1, l-KCNQ1 and KCNE1 gene
using QuickChange Kit (Stratagene). All the mutants were
characterised by restriction mapping and DNA sequencing.
An in-vitro transcription kit (Stratagene) was used to
synthesize cRNAs from WT and mutant s-KCNQ1 (SP6
kit), l-KCNQ1 (T7 kit) and KCNE1 (T7 kit) clones. Size,
integrity and quantity of cRNAs were verified by electrophoresis on formaldehyde / agarose gels together with
standards of known size and quantity. Kir 2.1 was used as
previously described [28].
2.2. Isolation of Xenopus laevis oocytes and
electrophysiology oocytes
Xenopus laevis were anaesthetised by immersion in
0.1% Tricaine (Sigma) for 30–60 min. After removal of
oocytes the Xenopus laevis were then humanely sacrificed
according to UK Home Office Schedule I guidelines.
Oocytes were prepared as previously described [28].
Injected oocytes were incubated in ND96 (in mmol / l, 96
NaCl, 2 KCl, 1 MgCl 2 , 5 HEPES, 1.8 CaCl 2 , pH 7.5 at
168C. Equimolar amounts of KCNQ1 and KCNE1 cRNAs
were injected. To study the dominant-negative effects,
different cRNA ratios between WT and mutant were used.
At a 1:1 ratio, equimolar amounts of cRNAs of mutant and
WT protein were injected. A 1:3 ratio was equivalent to
three times more mutant than wild type. Experiments were
analysed within a batch or pooled from a number of
batches as indicated.
Recordings were made 1–5 days after injection at room
temperature (22–258C) in a modified ND96 (mmol / l, 96
NaCl, 2 KCl, 2 MgCl 2 , 0.1 CaCl 2 , 5 HEPES, pH 7.6).
Currents were recorded with a standard two-electrode
voltage clamp technique using the TEV-200 (Dagan,
USA). Glass microelectrodes were filled with 3 M KCl and
had 0.3–0.8-MV resistance. pClamp5 software (Axon
Instruments) was used to generate voltage-clamp commands and collect experimental data. The membrane
potential was held at 280 mV between test pulses.
Currents were recorded during 3.5-s pulses from a holding
potential of 280 mV to test potentials ranging from 280 to
160 mV (10-mV intervals). Deactivating tail currents were
elicited by repolarisation to 250 mV. The inter-pulse
interval was 5 s. To determine the voltage-dependence of
the channel activation, oocytes were depolarised to potentials ranging from 280 1120 mV (10-mV intervals) for
3.5 s before repolarising to 250 mV. The voltage dependence of channel deactivation time constant was determined by repolarising the oocytes to potentials ranging
from 220 to 290 mV (10-mV intervals) for 5 s after a
3.5-s pulse to 160 mV.
Data analysis was carried out using pCLAMP 6.0.4,
Microsoft Excel 97 and Origin 6.0. Time constants were
determined by fitting currents to a single exponential decay
function. The voltage dependence of the channel activation
was determined by fitting the normalised amplitude of the
L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
672
Table 1
Summary of clinical details of heterozygotes studied
Mutation
Family
(proband
ascertained with
JLNS / RWS)
No. of
heterozygotes
studied
QTc (ms)
Family or personal
history of syncope or
sudden death (except
proband)
Refs. for
clinical
description
R243H
UK2T (JLNS)
3
430 (Mother)
429 (Father)
374 (Sister)
None
[9]
E261D
N14O (JLNS)
1
410 (Father)
None
[32]
R518X
N2S (JLNS)
1
Recorded as normal
N3S (JLNS)
Q530X
2 (Parents)
None
[31] (case 2)
Not available
a
None
[34,46]
a
None
[32]
N4O (JLNS)
1
Not available
UK5M (JLNS)
1
437 (Mother)
None
[32]
UK7B (RWS)
2
479 (Proband)
449 (Mother)
Syncope
No personal or other FH
This report
N7J (JLNS)
2 (Parents)
Recorded as normal
None
[1]
a
N6K (JLNS)
2 (Parents)
Not available
None
[34]
N14O (JLNS)
1 (Mother)
425–447
None
[32]
R594Q
UK5M (JLNS)
1
445 (Father)
None
[32]
1008delC
UK3C (JLNS)
1
428 (Mother)
None
[32]
572–576del
N2S (JLNS)b
1
Recorded as normal
None
[31] (case 2)
None
[32]
a
N4O (JLNS)
1
Not available
N10D (JLNS)
7
470 (Father)
430 (Mother)
430 (Great uncle)
420 (Second cousin)
450 (Second cousin)
500 (Second cousin)
No syncope
No syncope
No syncope
No syncope
No syncope
RWS with syncope
[34]
N5B (JLNS)
2
410 (Father)
470–540 (Mother)
None
[9]
N1H (JLNS)
2
400 (Father)
430 (Mother)
None
[31] (case 1), [9]
T59P/ L60P
(KCNE1)
UK1S (JLNS)
2 (Parents)
414 (Father)
418 (Mother)
None
[9]
L273F
UK8O(RWS)
4 (Siblings)
488
491
484
533
No
No
No
No
This report
1 (Parent)
Declined ECG
No syncope
syncope
syncope
syncope
syncope
Some families appear in the table twice as the JLNS proband was a compound heterozygote (e.g. N14O, N2S, N4O, UK5M).
a
Due to the long time since original diagnosis, no QTc intervals are available for these individuals although they had no cardiac symptoms.
b
For the mutation 572–576del, more than 26 heterozygotes from seven different families were studied in total [34].
peak tail currents ( y /y max ) versus test voltage potential (Vt )
with a Boltzmann function ( y /y max 5 1 /(1 1 exp[(V0.5 2
Vt ) /k])). Data were represented as mean6S.E.M. and
statistical comparisons were made using a two-tailed
Student’s t-test.
2.3. Family data
All homozygotes with JLNS were severely to profoundly deaf and had prolonged QTc intervals; two families
(UK80 and UK7B) were ascertained through syncope in
L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
the proband, and hearing was reported as normal in all
individuals in these two families. A series of criteria have
been presented for the diagnosis of long QT syndrome
[29]. As a generalisation a QTc greater than 440 ms in
males and greater than 460 ms in females is considered
prolonged in an individual who has symptoms or a family
history of sudden early cardiac death. For asymptomatic
individuals, QTc should be greater than 470 ms in order
for designation as ‘affected’ with long QT syndrome [30].
The family data together with relevant references [31–34]
are summarised in Table 1.
3. Results
The location and nature of the mutations in KCNQ1 and
KCNE1 examined in this study are shown in Fig. 1.
Site-directed mutagenesis was used to introduce these into
the respective cDNAs, and cRNA was synthesised and
injected into oocytes for electrophysiological study using
two-electrode voltage clamp (Methods).
3.1. Functional expression of currents
In agreement with previous studies [22,23], injection of
WT KCNQ1 cRNA alone led to the expression of low
levels of a rapidly activating potassium current (0.4060.05
mA at 160 mV, n56) and KCNE1 cRNA alone led to
low-level expression of a slowly activating potassium
selective current (1.2660.09 mA at 160 mV, n54). The
latter is due to the low level expression in Xenopus laevis
of an endogenous KCNQ1-like protein [22]. However,
coinjection of equimolar KCNQ1 and KCNE1 cRNAs led
to the prominent expression of a slowly activating potassium selective current (7.0260.48 mA at 160 mV, n511).
Mutant KCNQ1 cRNAs were coinjected together with WT
KCNE1 cRNA. None of the mutants produced any signifi-
Fig. 1. Mutations in KCNQ1 and KCNE1 used in this study [9,32].
673
cant current in the presence of WT KCNE1 (Fig. 2).
Furthermore, co-expression of the mutant KCNE1 (T59P/
L60P) and WT KCNQ1 cRNAs also did not induce any
significant current (Fig. 2).
3.2. Dominant negative effects
Potassium channels are tetrameric proteins [35] and thus
there is the potential for the presence of a single (or more)
mutant subunit in a tetramer to inactivate the function of
the remaining wildtype subunits. Consequently we examined the tendency for the above mutations to behave in
this way. Before undertaking these studies we first established the amount of KCNQ1 cRNA (together with
equimolar KCNE1 cRNA) that gave half-maximal current
expression. This ensured that we achieved measurable
currents but did not saturate the oocyte translation machinery. To further control for non-specific effects, the mutants
were injected together with a potassium channel (Kir 2.1)
from a totally distinct family [28].
KCNQ1 or KCNE1 mutants were coinjected at a 1:1
(Fig. 3) and 1:3 injection ratio (Fig. 4, WT:mutant).
E261D gave a pronounced reduction in current compared
to control (|65% at 1:1 ratio) and R243H a statistically
significant but more moderate effect (|25% at 1:1 ratio) at
both injection ratios. The KCNQ1 mutants (572–576del,
1008delC, R518X, Q530X and R594Q) did not give a
pronounced dominant negative effect at a 1:1 injection
ratio but did so at a 1:3 injection ratio. The KCNE1 mutant
T59P/ L60P did not significantly reduce the current when
co-expressed at either ratio with WT KCNE1 (in the
presence of KCNQ1). The dominant-negative effect of the
KCNQ1 mutants at high injection ratios is unlikely to be
due to non-specific effects as injection of the mutants
E261D or R243H at an amount equivalent to a 1:9 ratio
did not reduce Kir 2.1 current amplitudes. Current amplitudes measured at 2100 mV were 28.8061.21 mA (Kir
2.1, n55), 28.8460.48 mA (E261D1Kir 2.1, n55) and
28.9160.59 mA (R243H1Kir 2.1, n55), respectively.
One mechanistic possibility for the dominant negative
effect at a higher mutant to WT ratio is that KCNQ1
mutants could interact with WT KCNE1 protein. Thus
there would be less free KCNE1 protein to co-assemble
with WT KCNQ1 subunits to potentiate current. To test
this hypothesis, the amount of WT KCNE1 cRNAs was
increased four-fold, i.e the molar ratio between WT
KCNQ1 and KCNE1 became 1:4. Under these conditions a
prominent dominant negative effect with 572–576del was
still observed (Fig. 4C).
We compared our observations on the dominant negative
effects of these mutations in JLNS with a mutation
(L273F) we have studied in a family with RWS. We found
that at a 1:1 injection ratio there was a small but nonsignificant reduction in current (WT KCNQ11WT
KCNE154.2960.40 mA (n521), WT KCNQ11WT
KCNE11L273F53.7560.46 mA (n512), P.0.05). A
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L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
Fig. 2. Expression of KCNQ1 and KCNE1 mutations alone did not produce significant currents. (A) Currents recorded in oocytes expressing WT
KCNQ11WT KCNE1, R243H1WT KCNE1, E261D1WT KCNE1 and WT KCNQ11T59P/ L60P. Voltage protocol is shown in the inset. Oocytes were
depolarised for 3.5 s from a holding potential of 280 mV to test potentials ranging from 160 to 280 mV (10-mV intervals). Deactivating tail currents
were elicited by repolarisation to 250 mV. Note that the currents induced by coinjection of WT KCNQ1 and T59P/ L60P are similar to that induced by WT
KCNQ1 alone. (B) Current–voltage relationships of traces shown in A: j WT KCNQ11WT KCNE1, n R243H1WT KCNE1, s E261D1WT KCNE1,
d WT KCNQ11T59P/ L60P. (C) Bar graph of current amplitudes measured at 160 mV in oocytes co-expressing mutant KCNQ1 and KCNE1 in the
presence of WT KCNE1 and WT KCNQ1, respectively: Data are represented as mean6S.E.M. n numbers are in brackets above the bar graph. *P,0.05,
**P,0.01 in two-tailed Student’s t-test, analysed within the same batch of oocytes.
statistically significant dominant negative effect occurred
at a 1:3 injection ratio (WT KCNQ11WT KCNE15
2.060.0.38 mA (n58), WT KCNQ11WT KCNE11
L273F50.6660.06 mA (n56), P,0.01).
3.3. Effect of R243 H and other mutants on channel
kinetics
It was apparent from simple visual inspection of the
recordings that in coinjection experiments with WT
KCNQ1 (1equimolar KCNE1) and the R243H mutation
at both 1:1 and 1:3 ratios, there was a change in the
deactivation rate (Figs. 3A and 4A). We thus decided to
characterise this at a 1:1 injection ratio. It is apparent from
Fig. 5B that there is a significant rightward shift and
change in slope of the voltage activation curve. A kinetic
analysis revealed that the deactivation rate was increased
but the activation rate was not affected (Fig. 5C and D).
These findings prompted us to investigate whether the
other KCNQ1 or KCNE1 mutants affect channel kinetics.
None of the other KCNQ1 or KCNE1 mutants affected the
time constants of channel activation / deactivation (Table
2). Due to the small currents with the E261D mutation at a
1:1 injection ratio time constants were not determined.
3.4. Studies on l-KCNQ1
The experimental work described above was performed
on the short isoform of KCNQ1 (Methods). We performed
a comparable set of experiments on the long isoform of
L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
675
Fig. 3. Dominant negative effects at 1:1 injection ratio. (A) Currents recorded using the same voltage protocol as in Fig. 2 in oocytes expressing WT
KCNQ11WT KCNE1, WT KCNQ11572–576del1WT KCNE1, WT KCNQ11R243H1WT KCNE1, WT KCNQ11E261D1WT KCNE1 and WT
KCNE11T59P/ L60P1WT KCNQ1. Note that the deactivation tail current in the presence of R243H is faster than control. (B) Bar graph of current
amplitudes measured at 160 mV in oocytes co-expressing KCNQ1 and KCNE1 mutants at 1:1 ratio in the presence of WT KCNQ1 and WT KCNE1. Data
are analysed within the same batch of oocytes except for R243H and E261D in which they are pooled from three and two batches, respectively. Data are
represented as mean6S.E.M. *P,0.05, **P,0.01.
KCNQ1 examining expression, dominant negative effects
at 1:1 injection ratio and changes in kinetics with R243H,
and saw a similar pattern of behaviour (not shown).
4. Discussion
Our objective was to study the mechanism by which
disease-causing mutations in KCNQ1 and KCNE1 lead to
changes in I Ks and repolarisation abnormalities in patients
and carriers of JLNS mutations. In particular we sought to
understand why carriers of JLNS mutations are so rarely
symptomatic in contrast with carriers of RWS mutations
who are much more commonly symptomatic. We tried to
mimic the situation that is likely to occur in the patients
(homozygotes) and in the carriers (heterozygotes). The
simplest hypothesis is that RWS mutations cause a dominant negative effect in addition to the simple loss of
function, whereas JLNS mutations do not. Dominant
negative effects may arise because of the tetrameric nature
of potassium channels and reflect the ability of a single
mutant (or more) subunit in a tetramer to inactivate the
function of the three (or less) normal subunits. We used the
Xenopus laevis expression system for these studies. Whilst
it is a heavily used and productive technique, there are
some issues concerning its use. For example, KCNQ1 is
natively expressed at low levels [22,23], high levels of
cRNA injection can induce native anion currents [36] and
protein trafficking can be temperature dependent such as is
the case for mutant cystic fibrosis transmembrane conductance regulator [37,38].
All these mutations studied here inactivate the function
of KCNQ1 as a homomultimer. In addition, we coinjected
KCNQ1 with KCNE1 T59P/ L60P. Proline is a known
helix breaker and the occurrence of two such mutations in
the transmembrane domain might severely impair function.
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L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
Fig. 4. Dominant negative effects at 1:3 injection ratio. (A) Currents recorded using the same voltage protocol as in Fig. 2 in oocytes expressing WT
KCNQ11WT KCNE1, WT KCNQ11R243H1WT KCNE1, WT KCNQ11572–576del1WT KCNE1, and WT KCNE11T59P/ L60P1WT KCNQ1.
Note that the deactivation tail current in the presence of R243H is much faster than control. (B) Bar graph of current amplitudes measured at 160 mV in
oocytes co-expressing KCNQ1 and KCNE1 mutants at 1:3 ratio in the presence of WT KCNQ1 and WT KCNE1. (C) Increasing the injection dose of WT
KCNE1 cRNA did not affect the dominant-negative effect induced by 572–576del at 1:3 injection ratio. Data are represented as mean6S.E.M. *P,0.05,
**P,0.01, analysed within the same batch.
Indeed we failed to observe the characteristic enhancement
of current seen normally with KCNE1. Interestingly, the
KCNQ1 current was rapidly activating as occurs when it is
expressed in the absence of KCNE1. The failure to see a
dominant negative effect supports the idea that little stable
protein is synthesised.
The injection of mutant and WT cRNAs at 1:1 ratio
mimics the condition of heterozygous carriers with one
mutant and one WT allele. At 1:1 ratios, most JLNS
mutations did not cause a dominant negative effect. The
results suggest that carriers of the mutations observed in
six out of eight JLNS families (572–576del, 1008delC,
R518X, Q530X, R594Q, KCNE1 T59P/ L60P) would have
little additional impairment of I Ks current under normal
circumstances other than that due to the loss of a potentially functional allele. However, surprisingly at a 1:3 ratio
the KCNQ1 mutations did have a specific dominant
negative effect. It is possible that such effects might have
clinical sequelae. Indeed, one heterozygous carrier of
R518X, who presented with cardiac syncope diagnosed
with RWS had prolonged QTc although carriers were
asymptomatic in four other families with JLNS. Similarly
one heterozygous carrier of 572–576del had RWS with
cardiac syncope in an extended JLNS family. The only true
loss of function mutation was the KCNE1 T59P/ L60P in
which there was no dominant negative effect.
Two of these mutations did produce more pronounced
dominant negative effects. The strong dominant-negative
effect of E261D implies that heterozygous carriers may
have a long QT. One carrier had a normal QTc of 410 ms
although the extended family data are not available.
Though the R243H mutation was moderate in its effect we
L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
677
Fig. 5. The change of channel kinetics induced by R243H at 1:1 ratio. (A) Currents induced by injection of WT KCNQ11WT KCNE1 and WT
KCNQ11R243H (1:1)1WT KCNE1 using the voltage protocol shown on the top. Oocytes were repolarised from a 3.5-s pulse at 160 mV to potentials
ranging from 220 to 290 mV (10-mV interval) for 5 s. (B) Relative activation curves were determined using the voltage protocol shown in the inset.
Oocytes were depolarised to potentials ranging from 280 to 1120 mV (10-mV interval) for 3.5 s before repolarising to 250 mV. The current amplitudes
were measured at the beginning of the deactivating tail current at the indicated test potential and the data were normalised and fitted to a Boltzmann
function. WT (j): V0.5 528.0161.35 mV, k524.4261.20 mV (n513); R243H (h): V0.5 553.2561.44 mV, k530.2461.36 mV (n514). (C) Voltagedependent channel deactivation time constant was determined by fitting the tail currents induced by the voltage protocol shown in A to a single exponential
function. A faster deactivation rate in the presence of R243H was observed at all potentials (220 to 270 mV). WT (j): n514; R243H (h): n514. (D)
Voltage-dependent activation time constants were determined by fitting the currents induced by the voltage protocol shown in Fig. 2 to a single exponential
function. WT (j): n514; R243H (h); n514. Data are represented as mean6S.E.M. **P,0.01.
compared it with a known mutation L273F in RWS (also
present in a family we have studied). We found that the
dominant negative effect was at best equivalent to that
occurring with R243H. The functional effects of a number
of other mutations have been described in RWS and
generally they lead to a more pronounced dominant
negative effect [15].
The data therefore suggest some interesting conclusions.
The observation that the mutations may act in a dominant
negative manner at high injection ratios but not in 1:1
ratios with wild type may explain why most carriers of
JLNS mutations are asymptomatic but why occasionally
they may have symptoms. In our group of families whose
mutations were analysed because of presentation through a
proband with JLNS, only one obligate carrier of 45 had
symptoms (obligate carriers being parents of an index case
with JLNS or relatives who have been molecularly proven
to be heterozygous for the mutation segregating in a
family). If one includes the individuals heterozygous for
the R518X mutation but in whom the proband was
ascertained with RWS, this becomes two symptomatic
heterozygotes out of 46. This supports the concept of a
degree of myocardial repolarisation reserve [29,39]. In
other words it is possible to tolerate a degree of I Ks
reduction without clinical sequelae and it is likely that this
degree varies between individuals. Such ideas may explain
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L. Huang et al. / Cardiovascular Research 51 (2001) 670 – 680
Table 2
Time constants of channel activation and deactivation in the presence of KCNQ1 and KCNE1 mutants at 1:1 injection ratio
I ks channel type
Time constant of activation (s)
Time constant of deactivation (s)
WT KCNQ11WT KCNE1
R243H1WT KCNQ11WT KCNE1
WT KCNQ11WT KCNE1
1008delC1WT KCNQ11WT KCNE1
R518X1WT KCNQ11WT KCNE1
WT KCNQ11WT KCNE1
572–576del1WT KCNQ11WT KCNE1
Q530X1WT KCNQ11WT KCNE1
R594Q1WT KCNQ11WT KCNE1
T59P/ L60P1WT KCNQ11WT KCNE1
1.4760.09
1.5660.11
1.3160.10
1.3960.12
1.2360.12
1.3960.13
1.3360.11
1.2560.07
1.2960.09
1.1960.05
1.1460.05
0.5960.04
1.2860.07
1.1960.11
1.2560.12
2.2060.26
2.4360.78
2.8960.67
2.2660.29
3.0260.46
(n536)
(n529)
(n521)
(n512)
(n512)
(n512)
(n58)
(n57)
(n510)
(n511)
(n536)
(n529)**
(n521)
(n512)
(n512)
(n512)
(n58)
(n57)
(n510)
(n511)
Time constants of channel activation and deactivation were determined by fitting activating currents and tail currents at a single potential (160 and 250
mV, respectively) to a single exponential function. Time constants of channel activation and deactivation in the presence of E261D were not determined as
the currents were small. Data are represented as mean6S.E.M. (n5number of oocytes).
** P,0.01.
why some individuals are particularly predisposed to drug
induced long QT syndrome [40] and why we see a
spectrum of functional effects for these mutations in JLNS.
It is also consistent with the variable penetrance of some
mutations in RWS in some families [29]. It is clear that
there has to be total abolition of K 1 flux into the
endolymph for hearing loss to occur. What might the
cellular mechanisms of reserve be? It is quite possible that
there are variations in the current density of I Kr and I Ks
between individuals determined by genetic factors such as
promoter polymorphisms [41,42]. The spatial variation of
currents between different ventricular regions and the
response to adrenergic stimulation are also likely to be
important factors [29].
Our data also have implications for channel function. It
has been reported that there is a domain between amino
acids 590 and 620 in the KCNQ1 C terminus that
determines assembly [26]. Certainly both mutants that
showed a dominant-negative effect (E261D and R243H)
are missense mutations in S4–S5 linker with an intact C
terminus. Thus it is possible for the mutants to co-assemble with WT KCNQ1 subunits, whereas the other KCNQ1
mutants are non-sense (R518X, Q530X) and frameshift
mutations (572–576del, 1008del) with a truncated C
terminus or a missense mutation (R594Q) with an amino
acid change within the proposed assembly domain. Such
mutants are unlikely to co-assemble with WT KCNQ1.
However at higher injection doses, a dominant-negative
effect was found for all the KCNQ1 mutants. These
observations imply that another assembly domain exists
and the effect of 572–576del indicates that it lies before
S2–S3. It is established that voltage-gated channels of the
K v family have assembly domains in the N-terminus and
S1 [43].
Our data suggest an important role for the S4–S5 linker
in channel function and gating. E261D is a very conservative mutation but this mutant was non-functional and led to
a pronounced dominant negative effect. R243H was also
non-functional but when co-expressed with WT KCNQ1
and WT KCNE1 cRNAs, it caused a 125-mV rightward
shift of the midpoint (V0.5 ) in channel activation due to a
faster deactivation rate. Other mutations in the S4–S5
linker have been reported to change the gating of KCNQ1
[44] and some functional data has been reported on the
R243H mutation [45]. Our data suggest that some R243H
heteromultimers are functional.
In summary we have studied the functional effects of
eight JLNS mutations and our data indicate a spectrum of
functional effects for these mutations in keeping with the
variable but generally mild clinical effects on heterozygous
carriers. The fact that the functional effect may be altered
by the relative amounts of mutant and wild type protein
may suggest why predicting phenotype is not straightforward and why the effects of a mutation may differ
between individuals of the same or different families.
Acknowledgements
This work was supported by the British Heart Foundation (Dr Lingqian Huang), Medical Research Council (Dr
Maria Bitner-Glindzicz) and the Wellcome Trust (Dr
Andrew Tinker). We would like to thank for Dr Jess Tyson
for her help throughout the course of the study. We thank
Professor Mark Keating for the gift of s-KCNQ1 cDNA,
Dr Jacques Barhanin for the gift of l-KCNQ1 cDNA, Dr
Richard Swanson for the gift of KCNE1 and Dr E. Liman
for the gift of pGEMHE.
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