The nonsense mutations R518X-KCNQ1 and Q530X-KCNQ1 cause LQT1 (long-QT syndrome type 1) and result in a complete loss of IKs 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

The slow cardiac delayed outwardly rectifying potassium current (IKs) is responsible, alongside IKr, for late phase 2 and phase 3 repolarization of the human ventricular action potential. The IKs 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 IKs channel current density. Missense mutations can cause disease by disrupting the biophysical properties (gating characteristics) [5,6], trafficking or assembly of the IKs channel complex [711]. 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-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 IKs 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.

EXPERIMENTAL

Molecular biology

KCNQ1 (GenBank® accession number AF000571) was cloned into pcDNA3.1/Zeo(+) (Invitrogen) as described previously [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).

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 six-well (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 G-418 at various doses was added. In all experiments using PTC124, and in the comparative controls, the final concentration of DMSO 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 Lipofectamine™ 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 drug-free medium for 1 h.

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 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).

KCNQ1 topology, tertiary structure and location of the LQT1 nonsense mutations R518X and Q530X

Figure 1
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 IKs 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].

Figure 1
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 IKs 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].

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)2ATP (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 mΩ. 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 (V0.5) (steady-state 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.

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 IKs 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 IKs current and are therefore easily separated from any IKs current (Supplementary Figure S1). In contrast, KCNQ1+KCNE1 produced a slowly activating voltage-dependent outwardly rectifying current that is characteristic of IKs (Supplementary Figure S1). The current density and PTCD produced by KCNQ1+KCNE1 was significantly (P<0.05) larger than that seen for HEK-293 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 HEK-293 cells transfected with R518X+KCNE1 or Q530X+KCNE1 (Figure 2).

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; V0.5, the voltage, in mV, at which the channel is half-maximally activated.

Channel n Current density (+80 mV) (pA/pF) PTCD (+80 mV) (pA/pF) V0.5 (mV) Slope factor (mV) Activation t1/2 (+40 mV) (ms) Deactivation τ (+40 mV) (ms) 
KCNQ1+KCNE1 12 482.1±65.0* 144.5±19.4* 5.2±1.0 10.2±0.5 907.3±81.4 962.3±34.0 
KCNQ1+KCNE1+1 mM G-418 12 530.0±66.7* 161.2±21.5* 1.1±1.7 9.3±0.8 947.7±156.7 1013.6±58.9 
R518X+KCNE1 29.3±4.5† 3.8±1.0† ND ND ND ND 
R518X+KCNE1+1 mM G-418 10 282.5±55.2*† 118.2±24.1* 31.4±1.4† 17.1±0.9† 1837.8±132.4† 879.9±25.4 
Q530X+KCNE1 29.3±5.4† 3.6±1.6† ND ND ND ND 
Q530X+KCNE1+1 mM G-418 79.2±32.5† 29.3±14.5† 34.5±2.0† 18.5±1.4† ND 716.3±53.2† 
pcDNA3.1 33.6±8.6† 3.4±1.0† ND ND ND ND 
pcDNA3.1+1 mM G-418 25.5±9.7† 2.6±0.8† ND ND ND ND 
Channel n Current density (+80 mV) (pA/pF) PTCD (+80 mV) (pA/pF) V0.5 (mV) Slope factor (mV) Activation t1/2 (+40 mV) (ms) Deactivation τ (+40 mV) (ms) 
KCNQ1+KCNE1 12 482.1±65.0* 144.5±19.4* 5.2±1.0 10.2±0.5 907.3±81.4 962.3±34.0 
KCNQ1+KCNE1+1 mM G-418 12 530.0±66.7* 161.2±21.5* 1.1±1.7 9.3±0.8 947.7±156.7 1013.6±58.9 
R518X+KCNE1 29.3±4.5† 3.8±1.0† ND ND ND ND 
R518X+KCNE1+1 mM G-418 10 282.5±55.2*† 118.2±24.1* 31.4±1.4† 17.1±0.9† 1837.8±132.4† 879.9±25.4 
Q530X+KCNE1 29.3±5.4† 3.6±1.6† ND ND ND ND 
Q530X+KCNE1+1 mM G-418 79.2±32.5† 29.3±14.5† 34.5±2.0† 18.5±1.4† ND 716.3±53.2† 
pcDNA3.1 33.6±8.6† 3.4±1.0† ND ND ND ND 
pcDNA3.1+1 mM G-418 25.5±9.7† 2.6±0.8† ND ND ND ND 

Gentamicin and G-418, but not PTC124, induce readthrough of R518X and Q530X to produce full-length channel protein

Figure 2
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.

Figure 2
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.

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 G-418 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 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).

Extended dose range analysis of the readthrough activities of PTC124, gentamicin and G-418 on the nonsense mutation R518X

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.

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.

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 IKs-like characteristics (Figure 4 and Table 1). G-418 incubation did not induce the expression of IKs-like current from HEK-293 cells transfected with pcDNA3.1 (Figure 4 and Table 1). Although G-418 promoted the production of currents with IKs-like properties from R518X+KCNE1 and Q530X+KCNE1, the magnitude 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.

Patch-clamp analysis of the nonsense mutation suppressing activity of 1 mM G-418 on KCNQ1+KCNE1, R518X+KCNE1 and Q530X+KCNE1 channel function

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.

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.

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 pcDNA3.1-transfected cells (Figure 4b). Incubation with G-418 did not 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 IKs-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 IKs. In particular, we were keen to determine whether the amino acid substituted for the PTC during nonsense mutation suppression has effects on IKs 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 (V0.5) 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 V0.5 or rates of channel activation or deactivation of the wild-type channel (Figure 5 and Table 1).

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

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 (V0.5). The steady-state activation curves are fitted with a Boltzmann function (solid lines). (b) and (c) Rates of channel activation (activation t1/2) 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.

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 (V0.5). The steady-state activation curves are fitted with a Boltzmann function (solid lines). (b) and (c) Rates of channel activation (activation t1/2) 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 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 [21,2931]. 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 V0.5. Upon readthrough Q530X+KCNE1 also produced currents with significantly (P<0.05) rightward-shifted V0.5 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 (G-418) 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 IKs 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].

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 full-length 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 IKs channel function.

Abbreviations

     
  • 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

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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Supplementary data