Biochem. J. (2012) 443, 635–642 (Printed in Great Britain)
635
doi:10.1042/BJ20111912
Readthrough of long-QT syndrome type 1 nonsense mutations rescues
function but alters the biophysical properties of the channel
Stephen C. HARMER, Jagdeep S. MOHAL, Duncan KEMP and Andrew TINKER1
William Harvey Heart Centre, Barts & The London School of Medicine & Dentistry, Charterhouse Square, London EC1M 6BQ, U.K.
The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1
cause LQT1 (long-QT syndrome type 1) and result in a complete
loss of I Ks channel function. In the present study we attempted to
rescue the function of these mutants, in HEK (human embryonic
kidney)-293 cells, by promoting readthrough of their PTCs
(premature termination codons) using the pharmacological agents
G-418, gentamicin and PTC124. Gentamicin and G-418 acted to
promote full-length channel protein expression from R518X at
100 μM and from Q530X at 1 mM. In contrast, PTC124 did
not, at any dose tested, induce readthrough of either mutant.
G-418 (1 mM) treatment also acted to significantly (P < 0.05)
increase current density and peak-tail current density, at + 80 mV
for R518X, but not Q530X, to 58 +
− 11 % and 82 +
− 17 %
of the wild-type level respectively. However, the biophysical
properties of the currents produced from R518X, while similar,
were not identical with wild-type as the voltage-dependence
of activation was significantly (P < 0.05) shifted by + 25 mV.
Overall, these findings indicate that although functional rescue
of LQT1 nonsense mutations is possible, it is dependent on the
degree of readthrough achieved and the effect on channel function
of the amino acid substituted for the PTC. Such considerations
will determine the success of future therapies.
INTRODUCTION
length protein in mammalian cells [14]. Later, G-418 and another
aminoglycoside, gentamicin, were also found to be able to restore
the expression of full-length protein from nonsense mutations
in the CFTR (cystic fibrosis transmembrane conductance
regulator) [15]. A number of clinical trials have been conducted in
an effort to determine whether aminoglycosides, and gentamicin
in particular, are able to rescue the function of disease-causing
nonsense mutations [16]. Functional rescue has been seen for
patients with cystic fibrosis [17], but in general success has been
hampered by significant toxicity [16]. Other reagents have
been developed that are capable of nonsense mutation suppression. These include derivatives of aminoglycosides that have
been engineered to be less toxic, such as negamycin and NB54
[18,19], and compounds that are novel non-aminoglycosides such
as PTC124 (Ataluren), RTC13 and RTC14 [20,21]. PTC124 has
shown preliminary success in studies of cystic fibrosis [22], and
a Phase III trial is being conducted to assess efficacy in cystic
fibrosis associated with CFTR nonsense mutations [23].
In the present study, we decided to attempt to rescue the
function of two LQT1 nonsense mutations, R518X-KCNQ1 and
Q530X-KCNQ1, that cause JLNS and result in the production
of non-functional channels and a complete loss of the I Ks current
[11,24]. To do this we assessed and compared the abilities of three
readthrough-promoting agents (gentamicin, G-418 and PTC124)
to induce full-length protein expression and rescue the function
of the channels produced by R518X and Q530X.
The slow cardiac delayed outwardly rectifying potassium current
(I Ks ) is responsible, alongside I Kr , for late phase 2 and phase 3
repolarization of the human ventricular action potential. The I Ks
current is composed of a heteromeric combination of a tetramer of
pore forming α-subunits, KCNQ1, and between one and four (the
exact number remains the subject of debate) KCNE1 β-subunits
[1]. Mutations in KCNQ1 or KCNE1 can cause disorders of heart
rhythm and account for LQTS (long-QT syndrome) types 1 and
5 respectively [2]. Together, LQT1 and 5 mutations account for
∼ 50 % of all cases of hereditary LQTS [3]. LQTS is characterized
by a prolongation of the QT interval on an electrocardiogram. This
prolongation is caused by defects in repolarization which can lead
to the development of ventricular tachycardia and sudden death
[2,4].
Within LQT1 and LQT5 two different clinical syndromes
have been described. The first, RWS (Romano–Ward syndrome)
is autosomal dominant. The second, and rarer, JLNS (Jervell
Lange–Nielsen syndrome) is autosomal recessive, and these
individuals also have profound hearing loss and tend to have a
more severe clinical phenotype. In general, mutations in KCNQ1
and KCNE1 that cause LQTS act to reduce I Ks channel current
density. Missense mutations can cause disease by disrupting the
biophysical properties (gating characteristics) [5,6], trafficking
or assembly of the I Ks channel complex [7–11]. Nonsense
mutations can also cause LQT1 by introducing a PTC (premature
termination codon) in the open reading frame, which can result
in the production of a truncated protein or initiation of NMD
(nonsense-mediated mRNA decay) [2,12,13].
In 1985, it was first demonstrated that the aminoglycoside
antibiotics paromycin and G-418 were able to suppress the effect
of a nonsense mutation and result in the production of a full-
Key words: aminoglycoside, KCNE1, KCNQ1, Kv7.1, PTC124
(Ataluren), readthrough.
EXPERIMENTAL
Molecular biology
KCNQ1 (GenBank® accession number AF000571) was cloned
into pcDNA3.1/Zeo( + ) (Invitrogen) as described previously
Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; ECL, enhanced chemiluminescence; GFP, green fluorescent protein;
eGFP, enhanced GFP; HEK, human embryonic kidney; Hsp90, heat-shock protein 90; JLNS, Jervell Lange–Nielsen syndrome; LQTS/LQT, long-QT
syndrome; NMD, nonsense-mediated mRNA decay; PTC, premature termination codon; PTCD, peak-tail current density.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
636
Figure 1
S. C. Harmer and others
KCNQ1 topology, tertiary structure and location of the LQT1 nonsense mutations R518X and Q530X
(a) Schematic diagram of KCNQ1. The location of R518X and Q530X are highlighted. The anti-KCNQ1 antibody (α-KCNQ1) used in the experiments shown in Figures 2 and 3 is directed against a
distal C-terminal region of KCNQ1 (amino acid residues 661–676). (b) The I Ks channel is composed of a tetramer of pore forming KCNQ1 α-subunits and an unclear number of KCNE1 β-subunits
(see the text for details). The C-terminus of one of the four KCNQ1 α-subunits is also shown in order to demonstrate the location of R518X and Q530X in relation to the four α-helical domains
present in the C-terminus that are thought to be involved in directing tetramerization of KCNQ1 α-subunits [47]. (c) Location, sequence context and nucleotide base changes in KCNQ1 that result in
the generation of the LQT1 nonsense mutations R518X and Q530X [13].
[11]. R518X and Q530X were made by introducing the premature
stop codon generating mutation into the KCNQ1 sequence using
splicing by overlap extension PCR as described in [11]. KCNE1
(human synthetic sequence) was obtained from Dr Richard
Swanson (Merck and Co, Inc. Research Laboratories, West
Point, PA, U.S.A.) and was cloned into pcDNA3.1/Zeo( + ) on
EcoRI/NotI ends. All constructs were verified by automated
sequencing.
Chemicals
Gentamicin (G1914) was obtained from Sigma and G-418
(G0175) was from Melford. PTC124 (Ataluren) was manufactured on request by Exclusive Chemistry. Gentamicin and
G-418 were prepared in sterile Sigma-Grade water (catalogue
number W4502). PTC124 was prepared in cell-culture grade
DMSO (catalogue number D2650, Sigma).
in the culture medium was 1 %. At 24 h later, transfected cells
were washed once with ice-cold PBS + [PBS (pH 7.4) with
0.1 mM CaCl2 and 1 mM MgCl2 ] and scraped into 100 μl of
3× reducing SDS/PAGE loading buffer. Following harvesting,
lysates were sonicated briefly and incubated at 95 ◦ C for 10 min
to ensure complete denaturation.
For electrophysiological experiments, HEK-293 cells were
seeded the previous day at a low density on to 10 mm
coverslips (catalogue number 631-0170, VWR). The cells were
then transfected using LipofectamineTM 2000 (catalogue number
11668, Invitrogen) according to the manufacturer’s protocol. For
these transfections, 500 ng of each vector was co-transfected
with 100 ng of eGFP [enhanced GFP (green fluorescent protein)]
(pEGFP-N1; Clontech). At 4 h after the initiation of transfection
the cells were washed and fresh medium with or without 1 mM
G-418 was added and the cells were incubated for 24 h. Before
patching commenced, G-418-treated cells were incubated in drugfree medium for 1 h.
Cell culture and transfection
HEK (human embryonic kidney)-293 cells were cultured in
modified Eagle’s medium (catalogue number E15-825, PAA)
supplemented with 10 % FBS (fetal bovine serum) and
100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen).
For the analysis of nonsense mutation suppression by Western
blotting, HEK-293 cells were seeded at 70 % confluence in sixwell (35 mm) plates. The next day, cells were transfected using
TurboFECT (catalogue number R0531, Fermentas) according
to the manufacturer’s protocol. For these experiments, 750 ng
of each vector was transfected. At 4 h after the initiation of
transfection the cells were washed once and fresh medium
containing the presence or absence of PTC124, gentamicin or G418 at various doses was added. In all experiments using PTC124,
and in the comparative controls, the final concentration of DMSO
c The Authors Journal compilation c 2012 Biochemical Society
SDS/PAGE and Western blot analysis
Equal amounts of protein lysate were separated by SDS/PAGE.
Gels were then transferred on to PVDF membrane and blocked
for 1 h in PBS (pH 7.4) containing 5 % non-fat dried skimmed
milk powder. After blocking, membranes were incubated with
either anti-(KCNQ1 C-terminus) (Figure 1a) (residues 661–676)
(1:5000 dilution) (rabbit polyclonal; catalogue number APC-022,
Alomone) or anti-Hsp90 (heat-shock protein 90) (1:2000 dilution)
(rabbit polyclonal; catalogue number sc-7947, Santa Cruz
Biotechnology) antibodies for 2 h. To remove unbound primary
antibody, the membranes were washed three times with PBS for
5 min each. After washing, the primary antibody was detected
by incubating the blots with a HRP (horseradish peroxidase)conjugated goat anti-rabbit antibody (1:3000 dilution) (catalogue
Suppression of KCNQ1 nonsense mutations
637
Table 1 Electrophysiological and statistical analysis of the currents produced in HEK-293 cells, by KCNQ1, R518X and Q530X when co-expressed with
KCNE1, in the presence or absence of 1 mM G-418
For experimental details, please refer to Figures 4 and 5. Values are means +
− S.E.M. Statistical comparisons were performed using a one-way ANOVA with a Dunnett’s multiple comparison post hoc
test. *P < 0.05 compared with pcDNA3.1-transfected HEK-293 cells. †P < 0.05 compared with KCNQ1 + KCNE1-transfected HEK-293 cells. n , number of cells analysed; ND, not determined; V 0.5 ,
the voltage, in mV, at which the channel is half-maximally activated.
Channel
n
KCNQ1 + KCNE1
KCNQ1 + KCNE1 + 1 mM G-418
R518X + KCNE1
R518X + KCNE1 + 1 mM G-418
Q530X + KCNE1
Q530X + KCNE1 + 1 mM G-418
pcDNA3.1
pcDNA3.1 + 1 mM G-418
12
12
6
10
6
6
5
5
Current density
( + 80 mV) (pA/pF)
PTCD ( + 80 mV)
(pA/pF)
482.1 +
− 65.0*
530.0 +
− 66.7*
29.3 +
− 4.5†
282.5 +
− 55.2*†
29.3 +
− 5.4†
79.2 +
− 32.5†
33.6 +
− 8.6†
25.5 +
− 9.7†
144.5 +
− 19.4*
161.2 +
− 21.5*
3.8 +
− 1.0†
118.2 +
− 24.1*
3.6 +
− 1.6†
29.3 +
− 14.5†
3.4 +
− 1.0†
2.6 +
− 0.8†
number sc-2054, Santa Cruz Biotechnology) for 1 h. To
remove unbound secondary antibody, the membranes were
washed three times with PBS for 5 min. Blots were developed
using the ECL (enhanced chemiluminescence) Western blotting
chemiluminescent reagent kit (catalogue number RPN2108, GE
Healthcare) and the signal emitted was detected using Hyperfilm
ECL (catalogue number 28-9068-37, GE Healthcare).
Electrophysiology
Whole-cell voltage clamp recording was carried out using an
Axopatch 200B amplifier (Molecular Devices). All recordings
were made at room temperature (22 ◦ C). The intracellular
(pipette) solution contained: 150 mM KCl, 5 mM EGTA, 10 mM
Hepes, 2 mM MgCl2 , 1 mM CaCl2 and 5 mM (Na)2 ATP
(pH 7.2 with KOH). The extracellular (bath) solution contained:
150 mM NaCl, 5 mM KCl, 10 mM Hepes, 2 mM MgCl2 and
1 mM CaCl2 (pH 7.4 with NaOH). Transfected cells were
identified by epifluorescence (eGFP) and currents were recorded
at least 2 min after achieving the ‘whole-cell’ configuration. Cells
were kept at a holding potential of − 80 mV prior to recording.
To analyse the biophysical characteristics of currents present
in the transfected cells, stepped depolarizations were performed
from − 80 mV to + 80 mV in 10 mV increments for 6 s. Each
stepped depolarization was followed by a repolarizing pulse
back to − 20 mV for 2 s to measure resulting tail currents. In
between each depolarization the cells were held at − 80 mV.
Prior to recording, the series resistance was compensated by
at least 70 % using the in-built amplifier circuitry. When filled
with intracellular solution the pipette resistance was between
2 and 2.5 m. Electrophysiological data was analysed using
Clampfit (Molecular Devices) and Microcal Origin software. For
a detailed explanation of how current density, PTCD (peak-tail
current density), voltage-dependence of activation (V 0.5 ) (steadystate activation) and rates of channel activation and deactivation
were determined please refer to [25].
Data analysis
Data are expressed as means +
− S.E.M. Statistical analysis was
performed using GraphPad Prism. Statistical comparisons were
made using ANOVA with a Dunnett’s multiple comparison post
hoc test. The data were considered to be significantly different
when P < 0.05.
V 0.5 (mV)
Slope factor
(mV)
Activation t 1/2
( + 40 mV) (ms)
Deactivation τ
( + 40 mV) (ms)
5.2 +
− 1.0
1.1 +
− 1.7
ND
31.4 +
− 1.4†
ND
34.5 +
− 2.0†
ND
ND
10.2 +
− 0.5
9.3 +
− 0.8
ND
17.1 +
− 0.9†
ND
18.5 +
− 1.4†
ND
ND
907.3 +
− 81.4
947.7 +
− 156.7
ND
1837.8 +
− 132.4†
ND
ND
ND
ND
962.3 +
− 34.0
1013.6 +
− 58.9
ND
879.9 +
− 25.4
ND
716.3 +
− 53.2†
ND
ND
RESULTS
R518X and Q530X do not produce functional channels
The nucleotide mutations that generate R518X and Q530X and
location of the PTCs they introduce are shown in Figure 1. As reported previously [24], R518X + KCNE1 and Q530X + KCNE1
failed to produce a functional I Ks current (Supplementary
Figure S1 at http://www.BiochemJ.org/bj/443/bj4430635add.htm
and Table 1). The currents produced by R518X + KCNE1 and
Q530X + KCNE1 did not statistically (P > 0.05) differ from the
endogenous current present in HEK-293 cells when transfected
with empty pcDNA3.1 vector (Supplementary Figure S1 and
Table 1). The endogenous currents have previously been reported
[26] and are small in comparison with the current generated
by KCNQ1 + KCNE1. They also lack the slow activation
characteristics and tail current component of the I Ks current and
are therefore easily separated from any I Ks current (Supplementary
Figure S1). In contrast, KCNQ1 + KCNE1 produced a slowly
activating voltage-dependent outwardly rectifying current that
is characteristic of I Ks (Supplementary Figure S1). The
current density and PTCD produced by KCNQ1 + KCNE1
was significantly (P < 0.05) larger than that seen for HEK293 cells transfected with empty pcDNA3.1 vector, and also
possessed a tail current that was not present in empty vector
(pcDNA3.1)-transfected HEK-293 cells (Supplementary Figure
S1 and Table 1). In addition, the expression of full-length KCNQ1
channel protein could not be detected by Western blotting in HEK293 cells transfected with R518X + KCNE1 or Q530X + KCNE1
(Figure 2).
Aminoglycosides, but not PTC124, are able to restore expression
of full-length channel protein
Upon treatment with G-418 and gentamicin, but not PTC124,
expression of full-length channel protein from R518X + KCNE1
and Q530X + KCNE1 could be detected by Western blotting
(Figures 2a, 2b and 2c). For R518X + KCNE1, G-418
and gentamicin were able to induce readthrough of the
nonsense mutation at 100 μM and 1 mM respectively. For
Q530X + KCNE1, readthrough was less pronounced, with G418 and gentamicin promoting readthrough at 1 mM, but not
100 μM (Figures 2a and 2b). PTC124 did not appear to be able to
induce readthrough of R518X + KCNE1 or Q530X + KCNE1 at
100 μM (Figure 2c). It was not possible to analyse the readthrough
activity of PTC124 at 1 mM because a precipitate, which was toxic
c The Authors Journal compilation c 2012 Biochemical Society
638
Figure 2
S. C. Harmer and others
Gentamicin and G-418, but not PTC124, induce readthrough of R518X and Q530X to produce full-length channel protein
KCNQ1, R518X and Q530X were co-transfected with KCNE1 in HEK-293 cells. The cells were then incubated for 24 h with either no drug (-ve), or 100 μM or 1 mM of gentamicin, G-418 or PTC124.
Nonsense mutation suppression, and therefore full-length channel production, was analysed using an antibody that recognizes the last 16 amino acids of KCNQ1 (amino acid residues 661–676, see
Figure 1 for details). Gel loading was assessed using Hsp90 as a loading control. (a) The action of G-418 on nonsense mutation suppression. (b) The action of gentamicin (GENT) on nonsense
mutation suppression. (c) The action of PTC124 on nonsense mutation suppression. (d) The action of PTC124, gentamicin and G-418 on empty vector (pcDNA3.1)-transfected HEK-293 cells. Short,
medium and long exposure panels reflect the different film exposure times and enable a comparison to be made between the level of readthrough seen for R518X and Q530X and that seen for KCNQ1
in the presence/absence of the readthrough-promoting agents. The exposure times between the panels shown in (a), (b) and (c) are not directly comparable. All Western blots are representative of at
least three experiments. The molecular mass in kDa is indicated on the left-hand side.
to the cells (reduction in the anti-Hsp90 protein level), formed at
this concentration due to limited aqueous solubility (Figure 2d).
In comparison with the levels of channel protein expression
seen for KCNQ1 + KCNE1, the amount of full-length channel
produced from R518X + KCNE1 and Q530X + KCNE1, after
G-418 or gentamicin treatment, was low (Figures 2a and 2b). The
incubation of G-418 or gentamicin at 100 μM or 1 mM or PTC124
at 100 μM with KCNQ1 + KCNE1-transfected HEK-293 cells
did not appear to affect the level of channel expression (Figures 2a,
2b and 2c). Additionally, the incubation of G-418 or gentamicin
at 1 mM with pcDNA3.1 vector-transfected cells did not result
in the presence of full-length KCNQ1 channel protein or the
expression/up-regulation of a non-specific immunoreactive band
(Figure 2d). In case of any unusual dose–response characteristics,
and to further determine the nonsense suppression activity of
these agents, we also compared the readthrough activity of
G-418, gentamicin and PTC124 on the R518X nonsense mutation
over a wider range of concentrations [100 nM, 1 μM, 10 μM,
100 μM and 1 mM (not at 1 mM for PTC124)]. We were unable
to detect readthrough activity from PTC124 at any of the doses
tested, but both G-418 and gentamicin led to the production of
full-length channel protein at 100 μM and 1 mM (Figure 3).
Nonsense mutation suppression of R518X and Q530X can increase
current density
We next determined whether the channel proteins produced
from R518X and Q530X could form functional channels.
We performed whole-cell voltage clamp electrophysiological
recordings of HEK-293 cells transfected with KCNQ1 + KCNE1,
R518X + KCNE1, Q530X + KCNE1 and pcDNA3.1 that had
been incubated with 1 mM G-418 for 24 h. We chose to use
G-418 instead of gentamicin as it has been suggested to be more
potent at inducing readthrough [27]. When nonsense mutation
suppression was induced by G-418, R518X + KCNE1 and
Q530X + KCNE1 produced currents with I Ks -like characteristics
(Figure 4 and Table 1). G-418 incubation did not induce the
expression of I Ks -like current from HEK-293 cells transfected
with pcDNA3.1 (Figure 4 and Table 1). Although G-418
promoted the production of currents with I Ks -like properties
from R518X + KCNE1 and Q530X + KCNE1, the magnitude
c The Authors Journal compilation c 2012 Biochemical Society
Figure 3 Extended dose range analysis of the readthrough activities of
PTC124, gentamicin and G-418 on the nonsense mutation R518X
R518X + KCNE1 was transiently transfected into HEK-293 cells. At 4 h later PTC124, gentamicin
or G-418 at -ve, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM (not at 1 mM for PTC124) was
added to the cells and incubated for 24 h before harvesting of the cells. Nonsense mutation
suppression and therefore full-length channel production, was analysed using an antibody that
recognizes the last 16 amino acids of KCNQ1 (amino acid residues 661–676, see Figure 1
for details). Gel loading was assessed using Hsp90 as a loading control. Short and long
exposure times are included for each readthrough agent tested. Exposure times are not directly
comparable between different drug treatments. All Western blots are representative of at least
three experiments. The molecular mass in kDa is indicated on the left-hand side. α-Hsp90,
anti-Hsp90 antibody; α-KCNQ1, anti-KCNQ1 antibody.
of currents produced was very different. R518X + KCNE1
produced currents that had 58.5 +
− 11.4 % of the wild-type current
density at + 80 mV (Figure 4 and Table 1). R518X + KCNE1
also produced currents with significantly (P < 0.05) increased
PTCD at + 80 mV. In fact, the PTCD, after a depolarizing step
to + 80 mV, did not significantly (P > 0.05) differ from that seen
for KCNQ1 + KCNE1.
In comparison, after incubation with G-418, Q530X + KCNE1
produced much smaller currents with only 16.4 +
− 6.7 % of
the level of the wild-type current density. The increases
seen for current density and PTCD were variable and
did not quite reach significance (P > 0.05) when compared
with pcDNA3.1-transfected HEK-293 cells (Figure 4b and
Table 1). However, it was clear that a small amount of
readthrough of Q530X had occurred because the resulting currents
possessed small tail currents that were not seen in pcDNA3.1transfected cells (Figure 4b). Incubation with G-418 did not
Suppression of KCNQ1 nonsense mutations
639
Figure 4 Patch-clamp analysis of the nonsense mutation suppressing activity of 1 mM G-418 on KCNQ1 + KCNE1, R518X + KCNE1 and Q530X + KCNE1
channel function
(a) Representative traces of the currents produced by HEK-293 cells transfected with KCNQ1 + KCNE1, R518X + KCNE1, Q530X + KCNE1 and pcDNA3.1 when incubated for 24 h in the presence
of 1 mM G-418. The voltage protocol is shown in the inset and is described in detail in the Experimental section. A scale is also included (inset). (b) Representative traces of the currents produced by
pcDNA3.1- and Q530X + KCNE1-transfected HEK-293 cells when incubated for 24 h in the presence or absence of 1 mM G-418. Please note the difference in scale from (a). The voltage protocol
used is the same as that shown in (a). (c) Mean current–voltage relationships [current density (nA/pF)]. (d) Mean peak-tail currents (pA/pF). Values are means +
− S.E.M. The number of cells analysed
is indicated in Table 1.
significantly (P > 0.05) affect current density or PTCD from the
KCNQ1 + KCNE1 channel (Figure 4 and Table 1).
What are the functional properties of the channels produced upon
readthrough?
G-418 acted to significantly increase current density and PTCD
from R518X and also appeared to induce currents with I Ks -like
properties from Q530X (Figure 4). However, we wanted to assess
whether the biophysical properties of the currents generated upon
readthrough were the same as those seen for I Ks. In particular,
we were keen to determine whether the amino acid substituted
for the PTC during nonsense mutation suppression has effects on
I Ks function; this seemed particularly important given that even
conservative amino acid changes in KCNQ1 can result in profound
channel dysfunction [2,11]. Thus we compared the biophysical
properties of the currents produced on readthrough with those
of the wild-type KCNQ1 + KCNE1 channel (Figure 5 and
Table 1). For both R518X and Q530X, the currents produced had
voltage-dependences of activation (V 0.5 ) that were significantly
(P < 0.05) shifted towards depolarized potentials in comparison
with the wild-type channel (a shift of ∼ 25–30 mV) (Figure 5a and
Table 1). Furthermore, the rate of channel activation (activation
t1/2 ) was significantly (P < 0.05) lower for R518X + KCNE1
(Figure 5b and Table 1) than for wild-type current. It was not
possible to reliably measure the rate of channel activation for
Q530X + KCNE1 because of the lower levels of current and
the masking effect of the endogenous current. It was, however,
possible to measure the rates of channel deactivation (deactivation
τ ) for both mutants. Q530X, but not R518X, acted to significantly
(P < 0.05) increase the rate of channel deactivation in comparison
with the wild-type channel (Figure 5c and Table 1). G-418
incubation did not significantly (P > 0.05) alter the V 0.5 or rates
of channel activation or deactivation of the wild-type channel
(Figure 5 and Table 1).
c The Authors Journal compilation c 2012 Biochemical Society
640
S. C. Harmer and others
Figure 5 Biophysical properties of the currents generated by R518X +
KCNE1 and Q530X + KCNE1 upon induction of readthrough by 1 mM G-418
are not identical with those seen for the wild-type KCNQ1 + KCNE1 current
(a) Normalized voltage-dependent activation [SSA (steady-state activation)] curves (V 0.5 ). The
steady-state activation curves are fitted with a Boltzmann function (solid lines). (b) and (c) Rates
of channel activation (activation t 1/2 ) and deactivation (deactivation τ ) in response to changes
in voltage. Values are means +
− S.E.M. The number of cells analysed is indicated in Table 1.
DISCUSSION
In the present study, we attempted to rescue the function of
two LQT1 nonsense mutations by promoting readthrough of
their PTCs using three different pharmacological agents. We
found that G-418 and gentamicin, but not PTC124, are able
to promote the production of full-length channel protein and
this led to the presence of functional currents at the plasma
membrane. However, the degree of rescue that could be promoted
c The Authors Journal compilation c 2012 Biochemical Society
was not the same, with the rescue of R518X being much more
effective than that seen for Q530X. It is likely that the difference
in the degree of readthrough may be explained by the stop
codon each mutant contains. The success of readthrough is
inversely proportional to the strength of termination efficiency
and TAA is the strongest, TAG is intermediate and TGA is the
weakest [27]. Therefore since R518X possesses a TGA stop
codon and Q530X possesses a TAG stop codon (see Figure 1)
this probably explains why readthrough is greater for R518X
than Q530X. The amount of full-length channel protein produced
upon readthrough of R518X + KCNE1 following treatment with
gentamicin or G-418 was low in comparison with the level seen for
KCNQ1 + KCNE1. However, the level of PTCD produced upon
readthrough from R518X + KCNE1, after a depolarizing step to
+ 80 mV, was not significantly different (P > 0.05) to the level
seen for KCNQ1 + KCNE1 at 82 +
− 17 %. It is possible that this
discrepancy may be explained by regulation of the number of ion
channels present in the cell membrane. For KCNQ1 + KCNE1, in
comparison with R518X + KCNE1, more protein may be being
expressed, but a significant portion of this protein may be located
intracellularly. Indeed when KCNQ1 is fused with GFP at its
C-terminus (KCNQ1–GFP) and expressed (in conjunction with
KCNE1) in CHO (Chinese-hamster ovary) (CHO-K1) cells, a
significant proportion of the expressed protein is intracellularly
retained [9]. Additionally, we have found in previous studies that
the transfection of small amounts of KCNQ1 and KCNE1 cDNA
(40 ng per vector) into HEK-293 cells is sufficient to produce
fairly large currents [28], indicating that only relatively low levels
of channel protein expression are needed to generate substantial
levels of current density. In our hands, both G-418 and gentamicin
were equipotent at inducing full-length channel expression from
both mutants. In contrast, we were unable to see readthrough
activity with PTC124 at any of the concentrations we tested, from
100 nM to 100 μM. This finding was surprising in the light of
experimental work describing a maximal readthrough-promoting
activity for PTC124 of ∼ 3 μM [21,29–31]. The efficacy of
PTC124 is a controversial issue as two other recent reports
have also failed to promote readthrough using PTC124 [32,33].
Furthermore, it has been suggested that the activity of PTC124
may have been biased by the ability to interfere with the activity
of the reporter (firefly luciferase) used in the high-throughput
screening [34,35]. The success of Phase II clinical trials assessing
PTC124 in cystic fibrosis and Duchenne muscular dystrophy has
also been variable [23,36].
The rescue of nonsense mutation function by aminoglycosides,
in in vitro systems, has been reported for other ion channels
[15,37,38]. For LQT1 nonsense mutations, we were particularly
concerned that the channels produced upon readthrough would not
behave in the same way as the wild-type channel because even
very conservative mutations in KCNQ1, e.g. E261D, can have
severe effects on channel function and cause LQT1 [2,11]. It is
clear that aminoglycosides can reduce translational termination
fidelity, but the amino acid that replaces the premature stop
codon in mammalian cells remains unknown. In prokaryotes it
has been shown that tryptophan is incorporated at TGA stop
codons and that glutamine is incorporated at TAG and TAA
stop codons [39,40]. If this were also the case in mammalian cells
then R518X would become R518W and Q530X would revert to
the wild-type KCNQ1 sequence. When analysing the currents
produced by R518X + KCNE1 in the presence of G-418, we
found that they were similar, but not identical, to those produced
by wild-type KCNQ1 + KCNE1. We detected a slowing in the
rate of channel activation and a rightward depolarizing shift in
the V 0.5 . Upon readthrough Q530X + KCNE1 also produced
currents with significantly (P < 0.05) rightward-shifted V 0.5
Suppression of KCNQ1 nonsense mutations
and increased rates of channel deactivation, indicating that
readthrough did not produce channels with biophysical properties
identical with the wild-type for this mutant either. For both
mutants the biophysical differences in the properties of the
channels suggest that the amino acids substituted for the PTCs
have an adverse effect on channel function. Although beyond the
scope of the present study, and technically challenging, it would
be interesting to identify which amino acids are substituted for
the PTCs in these two mutants.
The concentrations of G-418 and gentamicin used in the
present study are approximately equivalent to those used in
studies that promoted the readthrough of nonsense mutations
in other ion channels such as CFTR [288 μM (G-418) and
837 μM (gentamicin)] [15], Kv1.5 [2 mM (gentamicin)] [37]
and HERG (human ether-a-go-go-related gene) [577 μM (G418) and 837 μM (gentamicin)] [38]. In these in vitro studies the
relative concentrations of aminoglycosides found to be effective
in promoting readthrough of nonsense mutations tend to be
high. Given the significant toxicity of aminoglycosides at higher
concentrations it is perhaps not surprising that their use in systemic
delivery applications has failed [16]. In contrast, applications
where aminoglycosides are administered locally, reducing the
potential for serious side effects, have been more successful, as
has been seen for CFTR [17]. For the successful rescue of LQT1
nonsense mutation function in vivo, gentamicin, or other agents,
would have to be administered systemically. However, on the basis
of the active concentrations shown in the present study, we feel
it is unlikely that clinically approved doses of gentamicin would
be capable of promoting enough readthrough to rescue function.
Indeed, the significant toxicity of aminoglycosides at active
concentrations has led to the development of aminoglycoside- and
non-aminoglycoside-based compounds that promote readthrough
at lower concentrations or with reduced or limited side effects,
such as NB54 [19] and RTC14 [20]. The development of these
new compounds may provide a way of rescuing the function of
LQT1 nonsense mutations, and other nonsense mutations, without
serious side effects.
It is important to note the limitations of the present study. These
experiments were performed in a heterologous cell system and
not in cardiac cells. However, it would be difficult to perform
such experiments in cardiac cells because of the presence of
endogenous I Ks currents. It is also difficult to extrapolate these
data to gauge potential success in in vivo systems. Transgenic
rabbit models have been developed, but were based on missense,
not nonsense, mutations [41]. Alternatively, human pluripotent
stem cell models that harbour specific nonsense mutations could
be used, and this strategy has recently been used to assess the
disease mechanisms of mutations in LQTS [42]. In the present
study, we have not investigated the role of NMD because the
cDNAs we use do not contain the genomic structure and splice
sites of the gene [43]. Previous studies have identified that NMD
plays an important role in regulating the success of readthrough
promotion [44]. The successful pharmacological rescue of R518X
and Q530X in vivo will therefore also be dependent on the
efficiency of NMD. If NMD is highly efficient it will not be
possible to produce enough full-length protein because of limited
mRNA transcript availability [44]. It is not possible to estimate
the availability of R518X and Q530X mRNA transcripts as NMD
efficiency has been shown to vary widely between tissue type and
individual [44]. Interestingly, the inhibition of NMD has been
shown to increase the effectiveness of gentamicin, by lowering the
effective dose needed, to promote readthrough of CFTR nonsense
mutations [44]. In the future, it may therefore be advantageous, in
an effort to reduce drug toxicity, to combine therapies that inhibit
NMD with those that promote readthrough [44].
641
Overall, we feel that the low and variable level of readthrough
seen for Q530X would be insufficient to rescue the cardiac
phenotype seen in these JLNS patients. In contrast, for R518X, the
amount of current produced was much larger and not that different
from wild-type. Interestingly, it has been suggested that as little as
10 % of the KCNQ1 current is sufficient to rescue hearing, and it
is thought that there is functional reserve in cardiac repolarization
[45,46]. However, functional rescue could be further complicated
by the altered biophysical properties of the currents generated.
In particular, the depolarizing shift in the voltage-dependence
of activation could act to reduce outward current during voltage
trajectory of the action potential, and the changes in channel
kinetics may affect rate adaptation of the current.
In conclusion, in the present study we have assessed the ability
of G-418, gentamicin and PTC124 to rescue the function of two
LQT1 nonsense mutations. We found that the aminoglycosides,
but not PTC124, were able to promote production of fulllength channels from both R518X and Q530X and significantly
(P < 0.05) increase current density for R518X, but not Q530X. In
addition, we found that the biophysical properties of the currents
produced by the mutants upon readthrough are not identical with
wild-type currents. In a clinical setting, the success of strategies
attempting to rescue the loss-of-function seen for LQT1 nonsense
mutations will therefore be highly dependent on the toxicity of
the readthrough promoting agent, nature and location of the stop
codon, and whether the amino acid substituted for the PTC has
an adverse effect on I Ks channel function.
AUTHOR CONTRIBUTION
Stephen Harmer, Jagdeep Mohal and Duncan Kemp performed the experiments. Stephen
Harmer and Andrew Tinker designed the experiments, analysed the data and wrote the
paper.
FUNDING
This work was supported by the British Heart Foundation [grant number
PG/09/026/27137].
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Biochem. J. (2012) 443, 635–642 (Printed in Great Britain)
doi:10.1042/BJ20111912
SUPPLEMENTARY ONLINE DATA
Readthrough of long-QT syndrome type 1 nonsense mutations rescues
function but alters the biophysical properties of the channel
Stephen C. HARMER, Jagdeep S. MOHAL, Duncan KEMP and Andrew TINKER1
William Harvey Heart Centre, Barts & The London School of Medicine & Dentistry, Charterhouse Square, London EC1M 6BQ, U.K.
Figure S1 Patch-clamp analysis of the currents produced upon transfection
of HEK-293 cells with the wild-type channel and the nonsense mutations
R518X and Q530X
Representative traces of the currents produced by HEK-293 cells 24 h after the transfection of
KCNQ1 + KCNE1, R518X + KCNE1, Q530X + KCNE1 or pcDNA3.1. The voltage protocol is
shown in the inset and is described in detail in the Experimental section of the main text. A scale
is also included (inset).
Received 26 October 2011/5 January 2012; accepted 6 February 2012
Published as BJ Immediate Publication 6 February 2012, doi:10.1042/BJ20111912
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society