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Cardiovascular Research 2006 70(3):466-474; doi:10.1016/j.cardiores.2006.02.006
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Copyright © 2006, European Society of Cardiology

Functional effects of a KCNQ1 mutation associated with the long QT syndrome

Inge R. Boulet, Adam L. Raes, Natacha Ottschytsch and Dirk J. Snyders*

Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp (UA), Universiteitsplein 1, 2610 Antwerp, Belgium

* Corresponding author. Department of Biomedical Sciences, University of Antwerp (UA), Universiteitsplein 1, 2610 Antwerp, Belgium. Tel.: +32 3 820 23 35; fax: +32 3 820 23 26. Email address: dirk.snyders{at}ua.ac.be

Received 13 September 2005; revised 24 January 2006; accepted 3 February 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective Long QT syndrome (LQTS) is an inherited disorder of ventricular repolarization caused by mutations in cardiac ion channel genes, including KCNQ1. In this study the electrophysiological properties of a LQTS-associated mutation in KCNQ1 (Q357R) were characterized. This mutation is located near the C-terminus of S6, a region that is important for the gate structure.

Methods and results Co-assembly of KCNE1 with the mutant Q357R elicited a current displaying slower activation compared to the wild-type KCNQ1/KCNE1 channels. The voltage dependence of activation of Q357R was shifted to more positive potentials. Moreover, a strong reduction in current density was observed that was partially attributed to the altered voltage dependence and kinetics of activation. The reduced current amplitude was also caused by intracellular retention of Q357R/KCNE1 channels as was shown by confocal microscopy. It indicated that the Q357R mutation disturbed protein expression by a trafficking or assembly problem of the Q357R/KCNE1 complex. To mimic the patient status KCNQ1, Q357R and KCNE1 were co-expressed, which revealed a dominant negative effect on current density and activation kinetics.

Conclusion The effects of the Q357R mutation on the activation of the channel together with a reduced expression at the membrane would lead to a reduction in IKs and thus in "repolarization reserve" under physiological circumstances. As such it explains the long QT syndrome observed in these patients.

KEYWORDS Ion channels; K-channel; Long QT syndrome

This article is referred to in the Editorial by A. Varro and J. Gy. Papp (pages 404–406) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The long QT syndrome (LQTS) is caused by an abnormally prolonged ventricular repolarization and is characterized by several symptoms including polymorphic ventricular arrhythmia known as "torsade de points" and ventricular fibrillation, sometimes leading to syncope or sudden death [1]. LQTS can be an autosomal dominant syndrome (Romano–Ward syndrome, RW) or an autosomal recessive disease, including in its phenotype also deafness (Jervell and Lange–Nielsen syndrome, JLNS) [2]. Mutations leading to congenital LQT have been identified in at least 7 genes, six of which are coding for ion channels [1,3], while Ankyrin B (LQT4) codes for a peripheral membrane anchor protein that binds to integral membrane proteins including cardiac sodium transporters [4].

Electrophysiologically, these mutations result in impaired sodium channel inactivation (LQT3) or cause a decrease in delayed outward rectifier potassium currents IK, both of which lead to a prolongation of the QT interval of the ECG of LQT patients [2]. IK is the major outward current responsible for ventricular repolarization [5]. At least three components of IK (IKr and IKs, IKur) have been identified in many mammalian species including humans. The delayed rectifier potassium channel KCNH2 (hERG) is responsible for the rapidly activating component IKr, and its dysfunction leads to LQT2 [6]. The homotetrameric KCNQ1 (KvLQT1) channel elicits a rapidly activating K+ current, but the co-assembly with KCNE1 (minK) results in a slow activation and an increase in current amplitude, generating the slowly activating delayed rectifier potassium current, IKs [7,8]. When studied with heterologous expression in Xenopus oocytes or mammalian cells, most missense mutations result in non-functional homotetrameric channels. In a heterotetrameric subunit composition, mimicking the patient status, a dominant negative effect is often observed [9–13]. In this study we characterized a mutation in KCNQ1 (Q357R, nucleotide change 1070 A>G) that was described by Chen et al. [14] in a 40-year old female patient who had a syncopal event. Her QTc was 0.43 s, which is within the normal range. She was treated with a β-blocker. The location of the mutation in the channel structure is disputed, as according to Ref. [14], the mutation is located in the S6 segment, but according to Refs. [3,15], this residue is situated in the intracellular C-terminal domain immediately after the S6 transmembrane segment. The functional consequences of the mutation were analyzed electrophysiologically and by confocal microscopy.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1 Molecular biology
hKvLQT1 in pSP64 was kindly provided by MT Keating (Harvard medical school, Boston, USA). It was subcloned into pBK/CMV by the use of Hind III and Sma I. The mutation was introduced with the QuickChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). After PCR based mutagenesis, a Xho I-Bgl II fragment containing the mutation was cut out of the PCR-amplified vector and ligated in hKCNQ1/PBK-CMV to replace the wild-type sequence. Double-stranded sequencing of the exchanged fragment and the adjacent sequence confirmed the presence of the desired modification and the absence of unwanted mutations. The plasmid DNA for mammalian expression was obtained by amplification in XL2 Bluescript cells (Stratagene), and then isolated from the bacterial cells with the endotoxin-free Maxiprep kit (Qiagen). The cDNA concentration was determined with UV absorption. hKCNE1 was expressed in a pBK-CMV expression vector.

2.2 Electrophysiology
CHO-K1 cells were cultured in Ham's F12 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The cells were transfected following the lipofection method using lipofectAMINE (Invitrogen). In the absence of KCNE1, 8 µg cDNA of the {alpha}-subunit (wild-type or mutant) was used. For the co-expression a mixture of 4 µg wild-type with 4 µg Q357R was used. In combination with KCNE1, the cells were always transfected with 4 µg cDNA of the {alpha}-subunit (wild-type or Q357R) and 2.5 µg of KCNE1. For the co-expression, 2 µg wild-type with 2 µg Q357R together with 2.5 µg of KCNE1 cDNA was used. 0.5 µg of GFP was co-expressed in each experiment. 8–24 h after transfection the cells were trypsinized and the GFP-fluorescent cells were used for analysis within 12 h. Current recordings were made with a Multiclamp-700B amplifier (Axon Instruments, Foster City, CA) in the whole cell configuration of the patch clamp technique. Experiments were performed at room temperature (20–23 °C); current recordings were low pass-filtered and sampled at 1–10 kHz with a Digidata 1322A data acquisition system (Axon Instruments). Command voltages and data storage were controlled with pClamp8 software (Axon Instruments). Patch pipettes were pulled from 1.2-mm borosilicate glass capillaries (World Precision Instruments, inc., Sarasota, Florida) with a P-2000 puller (Sutter Instruments, Novato, CA). After heat polishing, the resistance of the patch pipettes was < 3 M{Omega} in the standard extracellular solution.

The cells were perfused continuously with a bath solution containing 130 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, adjusted to pH 7.35 with NaOH. The intracellular solution used contained 110 mM KCl, 5 mM K4BAPTA, 5 mM K2ATP, 1 mM MgCl2, 10 mM HEPES and was adjusted to pH 7.2 using KOH. Junction potentials were zeroed with the filled pipette in the bath solution. The remaining liquid junction potential was estimated to be 1.7 mV and was not corrected [16]. After achieving a gigaohm seal, the whole cell configuration was obtained by suction. Capacitive transients were elicited by applying a –  10 mV voltage step to determine the capacitive surface area, access, and input resistance. The access resistance varied from 3 to 9 M{Omega} without compensation and was below 3 M{Omega} after whole cell compensation. Experiments were excluded from analysis if the voltage errors originating from series resistance were greater than 5 mV.

2.3 Data analysis
The holding potential was –  80 mV. The interpulse interval was at least 15 s. The current recordings were fitted with a single exponential function to determine the time constants for activation and deactivation. The voltage dependence of channel activation was fitted with a Boltzmann equation: y=1/1+exp (–  (E – V1/2)/k)), in which k represents the slope factor, E is the applied voltage, and V1/2 is the voltage at which 50% of the channels are activated. Results are expressed as mean±SEM. For the statistical analysis of the current densities, an unpaired t-test was performed.

2.4 Confocal imaging
WT and mutant KCNQ1 were tagged with GFP at their carboxy terminus. CHO-K1 cells were grown on cover slips and transfected using lipofectamine. For transfection 2 µg of the WT KCNQ1-GFP or mutant Q357R-GFP cDNA and 1 µg KCNE1 cDNA was used. The co-expression was performed both with the WT KCNQ1-GFP tag (1 µg) combined with the untagged mutant Q357R (1 µg) and KCNE1 (1 µg) (Q1-GFP+Q357R+E1), or with the mutant Q357R-GFP tag (1 µg) combined with the untagged WT KCNQ1 (1 µg) and KCNE1 (1 µg) (Q1+Q357R-GFP+E1). 48 h after transfection, confocal images were obtained on a Zeiss CLSM 510, equipped with an argon laser (excitation, 488 nm) for the visualization of GFP. The prolongation of the recovery time after transfection (compared to cells used for electrophysiology) resulted in a more clear morphology of the cells. The current density is not affected by longer recovery times (data not shown) allowing the comparison of confocal images with electrophysiological data.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1 Kinetic properties of the WT and Q357R
In the topology of the KvLQT1(KCNQ1) subunit, the residue Q357 is located at the end of the S6 transmembrane domain [14] or one of the first residues immediately beyond the S6 transmembrane domain [3,15]. The WT or mutant cDNA was transiently transfected into CHO-K1 cells in the absence or in the presence of the regulatory subunit KCNE1. KCNQ1 elicited an outward rectifying potassium current characterized by an exponential activation time course and a threshold of activation of approximately –  40 mV. Upon repolarization, a slow deactivation process was preceded by a transient increase in the current (a "hooked tail") resulting from a rapid recovery from inactivation [8] (Fig. 1A). In the absence of KCNE1, the Q357R mutation induced a marked change of the KCNQ1 current (Fig. 1B). The time course of activation of homotetrameric Q357R channels was slower compared to WT channels and the threshold of activation was slightly shifted to more positive potentials (Fig. 1B). To determine the voltage dependence of activation, the peak amplitudes of normalized tail currents recorded at –  40 mV were plotted as a function of the prepulse potential. The midpoints of activation were calculated by fitting with the Boltzmann equation (Fig. 1C). For the homotetrameric Q357R channel, a small but not significant shift was observed compared to the KCNQ1 channels. The time constants of activation and deactivation were obtained by fitting an exponential function to the activating or deactivating currents, respectively. While the activation time constants for Q357R were increased by 10-fold, the deactivation time constants were essentially unchanged (Fig. 1D). The current–voltage relationships showed that the current density for Q357R channel was decreased by nearly 2-fold compared to KCNQ1 although this reduction was not statistically significant (Fig. 2A).


Figure 1
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  		Fig. 1 Representative current traces for CHO-K1 cells transfected with (A) KCNQ1 (8 µg) or (B) Q357R (8 µg). The voltage protocol is shown on top of panel A: cells were clamped at a holding potential of –  80 mV, and 800 ms pulses of voltages from + 80 to –  60 mV were imposed in steps of –  10 mV. Tail currents were recorded by stepping to –  40 mV for 1280 ms. (C) Voltage dependence of activation: activation curves were obtained by plotting the normalized tail currents as a function of the pre-pulse potential. The midpoints of activation were obtained by fitting with the Boltzmann equation (solid lines). No significant difference in midpoint potential between KCNQ1 (filled squares) (V1/2= –  0.3±2.5, k=16.0±1, n=7) and Q357R (open squares) (V1/2=+ 7.3±2.4, k=14.5±1.2, n=6) was observed. (D) Activation and deactivation kinetics of KCNQ1 and Q357R: time constants derived from mono-exponential fits of the raw current traces were plotted as a function of membrane potential. The activation kinetics for the mutant channel were slowed compared to the WT channel, no difference in deactivation kinetics was observed.

 

Figure 2
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  		Fig. 2 Current–voltage relationship for recordings as shown in Figs. 1 and 3Go. The current amplitudes±S.E.M were measured at the end of the activation voltage step for homotetrameric KCNQ1 (filled squares) (n=23) and Q357R (open squares) (n=15) channels and after a 5 s depolarizing pulse for (A) the KCNQ1/KCNE1 (filled circles) (n=16) and (B) Q357R/KCNE1 (open circles) (n=8) channels. The current densities were calculated by dividing the current amplitudes by the cell capacity, and plotted as a function of the pulse potential. The Q357R mutation caused a reduction in current density for the Q357R/KCNE1 channel complex (p<0.001) and the homotetrameric Q357R channels (p=0.06). However, the reduction for the Q357R/E1 complex was far more pronounced.

 
Co-expression of KCNQ1 with KCNE1 markedly modified the characteristics of the currents. The KCNQ1/KCNE1 complex activated more slowly compared to KCNQ1 alone (Figs. 1A, 3AGo) with an activation threshold of approximately –  20 mV. The current amplitude of KCNQ1/KCNE1 channels was larger (8-fold) than that of the homotetrameric KCNQ1 channels (compare Fig. 1A with Figs. 3A and 2AGo with B). The co-expression of the mutant Q357R subunit with KCNE1 elicited a current with an even slower time constant of activation compared with the KCNQ1/KCNE1 complex, with a threshold for activation that was shifted by almost + 20 mV (Fig. 3B). The activation curve of the Q357R/KCNE1 channel was equally shifted by + 20 mV compared to KCNQ1/KCNE1 (Fig. 3C). Both the homotetrameric Q357R channel and the current generated by the Q357R/KCNE1 complex showed a slower rate of activation (Figs. 1D, 3DGo). In the presence of KCNE1, there was no obvious effect of the Q357R mutation on the deactivation kinetics (Fig. 3D). The current–voltage relationships of KCNQ1 and Q357R in the presence of KCNE1 are shown in Fig. 2B. For Q357R/KCNE1 channels a significant decrease in current density was observed compared to the KCNQ1/KCNE1 channels (Fig. 2B).


Figure 3
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  		Fig. 3 (A) Representative current traces of CHO-K1 cells transfected with KCNQ1 (4 µg)+KCNE1 (2.5 µg) or (B) Q357R (4 µg)+KCNE1 (2.5 µg) channel. The voltage protocol is shown on top of panel A: cells were clamped at a holding potential of –  80 mV and 5 s (KCNQ1/KCNE1) or 10 s (Q357R/KCNE1) pulses of voltages from + 60 to –  40 mV were imposed in steps of –  10 mV. Tail currents were recorded by stepping to –  40 mV for 5 s. Inset shows trace at + 60 mV for a 5 s depolarizing step. (C) Activation curves fitted with the Boltzmann equation (solid lines): the activation curve of the mutant complex Q357R/KCNE1 (open circles) (V1/2=+ 47.6±1.6, k=11±1, n=7) showed a significant shift (p<0.001) compared to KCNQ1/KCNE1 (filled circles) (V1/2=+ 28.0±3.1, k=14.3±1.4, n=5). (D) Activation and deactivation kinetics of KCNQ1 and Q357R in the presence of KCNE1: time constants derived from mono-exponential fits of the raw current traces were plotted as a function of membrane potential. The activation kinetics for the mutant channel combined with KCNE1 were slowed compared to the KCNQ1/KCNE1 channel, no difference in deactivation kinetics was observed.

 
3.2 Co-expression of Q357R and KCNQ1 in the absence and presence of KCNE1
Fig. 4A shows the raw current traces for the co-expression of KCNQ1 and Q357R in a 1:1 ratio. The resulting channels displayed a small but significant shift towards positive potentials, comparable to the one observed for homotetrameric Q357R (Figs. 1C, 4CGo). The activation kinetics for the co-expression of KCNQ1/Q357R were intermediate compared to the KCNQ1 and Q357R channels while deactivation kinetics remained unchanged (Fig. 4B). The current density of the heterotetrameric complex KCNQ1/Q357R was reduced almost 2-fold compared to KCNQ1, but this was not statistically significant (Fig. 4D). These altered biophysical properties support the heterotetrameric nature of the channels formed by the co-expression.


Figure 4
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  		Fig. 4 Co-expression of KCNQ1 and Q357R subunits. (A) Representative current traces of CHO-K1 cells transfected with equal amounts (4 µg) of KCNQ1 and Q357R subunits. Cells were clamped at a holding potential of –  80 mV, and 800 ms pulses of voltages from + 80 to –  60 mV were imposed in steps of –  10 mV. Tail currents were recorded by stepping to –  40 mV for 1280 ms. (B) Activation and deactivation kinetics of the heterotetrameric channels. Time constants were derived from mono-exponential fits of the raw current traces and were plotted as a function of membrane potential. For the heterotetrameric KCNQ1/Q357R channels (semi-filled squares) the activation kinetics were slowed while deactivation was unaltered compared to KCNQ1 (filled squares). (C) Activation curves for the heterotetrameric channels fitted with the Boltzmann equation (solid lines). The heterotetrameric channel KCNQ1/Q357R (V1/2=+ 10.7±3, k=19.1±1.3, n=6) showed a small but significant shift (p<0.05) in voltage dependence compared to the WT channel channels (V1/2= –  0.3±2.5, k=16.0±1, n=7). (D) Current densities were obtained from recordings like in Fig. 1A (KCNQ1: n=23) and Fig. 4A (Q1/Q357R: n=14). Current amplitudes±S.E.M were determined at the end of a 800 ms depolarizing pulse, normalized to the cell capacity and plotted as a function of the pulse potential.

 
To mimic the heterozygous status of the patient, we co-expressed KCNQ1, Q357R and KCNE1 (1:1:1.25 ratio) (Fig. 5A). The time constants of activation for KCNQ1/Q357R/KCNE1 were slower than for KCNQ1/KCNE1 but faster than for Q357R/KCNE1, while the kinetics of deactivation were unaltered (Fig. 5B). The voltage dependence of activation was shifted towards positive voltages compared to the KCNQ1/KCNE1 complex although to a lesser extent than homotetrameric Q357R/KCNE1 (Fig. 5C). The current density for the heterotetrameric complex KCNQ1/Q357R/KCNE1 was significantly reduced (~60%) compared to the KCNQ1/KCNE1 (Figs. 5D, 6AGo). This would be consistent with a reduced trafficking of channel complexes. The voltage dependence of activation was shifted towards positive voltages and this was associated with a pronounced slowing of the activation for both Q357R/KCNE1 and KCNQ1/Q357R/KCNE1. Evidently, these alterations themselves could cause the reduced current density because the current amplitude was measured in an isochronal fashion (at the end of the 5 s pulse to + 60 mV). Therefore, the current density was also obtained at + 60 mV at the time point corresponding to one time constant for both the mutant Q357R/KCNE1 complex and the co-expression KCNQ1/Q357R/KCNE1. With this correction for the activation kinetics, the current densities were also significantly reduced both for the homotetrameric Q357R/KCNE1 and for the heterotetrameric KCNQ1/Q357R/KCNE1 channels (Fig. 6B) suggesting that the presence of a Q357R subunit in a channel complex caused a reduction of the plasma membrane expression of the channel complex.


Figure 5
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  		Fig. 5 Co-expression of KCNQ1 and Q357R subunits in the presence of KCNE1. (A) Representative current traces of CHO-K1 cells transfected with KCNQ1 (2 µg)+Q357R (2 µg)+KCNE1 (2.5 µg) subunits. Cells were clamped at a holding potential of –  80 mV, and 5 s pulses of voltages ranging from + 60 to –  40 mV in steps of –  10 mV were imposed. Tail currents were recorded by stepping to –  40 mV for 5 s.(B) Activation and deactivation kinetics of the heterotetrameric channels. Time constants derived from mono-exponential fits of the raw current traces were plotted as a function of membrane potential. To accurately determine the time constants of activation, the activation protocol was also recorded with 10 s pulses of voltages ranging from + 60 to –  40 mV in steps of –  10 mV. For the heterotetrameric channel KCNQ1/Q357R/KCNE1 (semi-filled circles) the activation kinetics were slowed and no difference in deactivation kinetics was observed compared to KCNQ1/KCNE1 (filled circles). (C) Activation curves for the heterotetrameric channels in the presence of KCNE1 were fitted with a Boltzmann equation (solid lines). The heterotetrameric channel KCNQ1/Q357R/KCNE1 (V1/2=+ 35.5±2.4, k=14.2±0.7, n=5) showed an intermediate shift in voltage dependence compared to the KCNQ1/KCNE1 channel and the Q357R/KCNE1 channel (open circles). (D) Current densities were obtained from recordings as shown in Fig. 3A (Q1/E1: n=16), 3B (Q357R/E1: n=8) and 5A (Q1/Q357R/E1: n=15). Current amplitudes±S.E.M were determined after a 5 s depolarizing pulse, normalized to the cell capacity and plotted as a function of the pulse potential.

 

Figure 6
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  		Fig. 6 (A) Bar graph showing the current density obtained from the peak current after a 5 s pulse at + 60 mV. Compared to KCNQ1/KCNE1 the current densities of Q357R/KCNE1 and KCNQ1/Q357R/KCNE1 were significantly different (*p<0.001). (B) Bar graph showing the current density obtained from the peak current at + 60 mV after one time constant to compensate for the slower activation kinetics. The current densities were significantly different from KCNQ1/KCNE1 (*p<0.001 for Q357R/KCNE1; **p<0.05 for KCNQ1/Q357R/KCNE1).

 
3.3 Subcellular localization of KCNQ1 and Q357R
To investigate the effect of the Q357R mutation on expression of the channel at the level of the plasma membrane, confocal microscopy of GFP tagged subunits was performed. Typical images for the subcellular localization of the KCNQ1-GFP and Q357R-GFP channels are shown in Fig. 7. In agreement with the reduced current density, the surface expression of the Q357R-GFP channels was decreased compared to that of wild-type KCNQ1-GFP channels and was associated with more intracellular retention (Fig. 7). The heterotetrameric channel KCNQ1/Q357R/KCNE1 also showed less membrane associated fluorescence compared to the homotetrameric KCNQ1/KCNE1 channel (Fig. 7). These results further indicate that the Q357R mutation leads to increased intracellular retention of the mutant channel.


Figure 7
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  		Fig. 7 Sub-cellular localization of KCNQ1 and Q357R both with KCNE1, determined by confocal microscopy. The subunits were tagged with GFP at the C-terminal end. Scale bar indicates 10 µm. Note that the clear membrane localization of the KCNQ1/KCNE1 channels is not observed with Q357R/KCNE1 or KCNQ1/Q357R/KCNE1 (the latter mimicking the patient status). To exclude the possibility that the GFP fusion itself affected the trafficking of the channel, the co-expression was performed both with the WT KCNQ1-GFP combined with the untagged mutant Q357R and KCNE1 or the Q357R-GFP combined with the untagged WT KCNQ1 and KCNE1. Both these co-expressions generated similar results.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
Co-assembly of the KCNQ1 (KvLQT1) {alpha}-subunit with its accessory subunit KCNE1 (minK) recapitulates the properties of the cardiac IKs current [7,8]. Since the IKs current is implicated in forming a "repolarization reserve" in the heart when the action potential duration (APD) is prolonged [5,17], the reduction of the IKs current due to mutations in the KCNQ1 gene could result in inherited LQT1 syndrome with high risk for ventricular tachycardia and ventricular fibrillation [18,19].

Many KCNQ1 mutations are located in the transmembrane core of the protein, such as the S2–S3 and S4–S5 loops, the S6 transmembrane segment or the pore domain. In addition, multiple LQT1 mutations are located in the C-terminal part of the channel [3,20]. Most of the mutated residues are highly conserved and these mutations typically result in a total loss of function in the homotetrameric state. The location of the Q357R mutation is somewhat ambiguous as some authors located it in S6 [14] while others placed it in the C-terminus [3,15]. In any case, the sequence 351–360 is as conserved as the rest of the S6 sequence. We examined the electrophysiological properties of the Q357R mutation in KCNQ1 by heterologous expression in CHO-K1 cells and by using whole cell voltage clamp.

In contrast to many previously described and characterized mutations in KCNQ1 [9–13], the Q357R mutation did not cause a total loss of function, but induced a functional channel with marked changes in activation kinetics and voltage dependence. The current generated by the homotetrameric Q357R channel showed a slower rate of activation compared to the WT KCNQ1 homotetramers. This slowing of the activation rate caused by the Q357R mutation was also present in co-expression with KCNE1; Q357R with KCNE1 activated more than 2-fold slower than KCNQ1/KCNE1 and the voltage dependence of activation for the mutant complex Q357R/KCNE1 was shifted to positive potentials. The synergistic nature of these two effects would lead to a reduction of the current.

To mimic the heterozygous in vivo situation with both the WT and mutated alleles co-existing in the same individual, co-expression experiments were performed in which equal amounts of WT and mutated DNA were transfected with or without KCNE1. Under these conditions we still observed a decreased current density compared to the WT complex, suggesting a dominant negative effect of the Q357R subunit in the context of the heterotetrameric channel. Several studies have reported the dominant negative suppression of IKs by KCNQ1 mutants [11]. Most of these mutations caused a total loss of function in the homotetrameric state and resulted in a dominant negative effect in co-expression with the WT subunit [9,10,12,13,15,18,21]. This dominant negative effect consisted of a reduction in current density although the kinetic properties of the resulting current largely remained unaffected. However, LQT1 mutations that did not cause a loss of function but affected the gating properties of the IKs channel have been described previously [15,20], including several mutations in the C-terminus of KCNQ1 and one mutation in S4. In combination with KCNE1, these mutations shifted the voltage dependence of activation, affected the deactivation kinetics and reduced the current density. Furthermore, in co-expression with the WT subunit, the mutations caused a dominant negative suppression of the KCNQ1 function. The Q357R mutation also displayed a reduced current density compared to KCNQ1 that could only partially be explained by the positive shift in voltage dependence of activation and the changed activation kinetics of the mutant complex Q357R/KCNE1. After correction for the reduced rate of activation, a significant reduction in current density remained, likely caused by a reduced protein expression at the level of the plasma membrane as could be demonstrated by confocal microscopy. At present it is unclear whether trafficking or assembly problems of the Q357R/E1 complex underlie the altered current density. Indeed, we can not exclude that the association with KCNE1 was disturbed by the mutation leading to a reduced surface expression. However, the latter is unlikely as the typical slowing of the activation as observed with KCNE1 is still present. Failure of trafficking is an increasingly recognized mechanism for cardiac ion channel disease [22]. LQT2 mutations resulting in hERG trafficking defects are well described [22–24]. For KCNQ1, the C-terminal end of the channel was identified as an important region for channel processing [24]. It was reported that a number of mutations in KCNQ1 were retained in the endoplasmic reticulum and that these mutants displayed a dominant negative effect on the WT channel [25–28]. While some of them were located in the C-terminus and confirming its role [26,28], some were located outside the C-terminus including an in frame deletion DeltaS276 in S5 [26], and some missense mutations in the S4–S5 linker [27]. It demonstrates that the intracellular C-terminal end of the channel is important in trafficking but shows that other parts of the channel are also involved. While most of these mutations caused a severe impairment of the trafficking of the homotetrameric mutant channel, leading to a total loss of function, the Q357R mutation was less severe as the channel still induced current in a heterologous expression system. Furthermore, the Q357R mutation is the first mutation with a trafficking defect in the highly conserved end of S6 (on the junction of S6 with the intracellular C-terminus).

In cardiac myocytes, the IKs current is modulated by the sympathetic nervous system [29,30]. LQT1 mutations in the C-terminus of KCNQ1 have been shown to diminish the β-adrenergic regulation of the IKs current by disturbing the binding domain of the A-kinase-anchoring protein yotiao. However, as this domain is located at positions 588–616 in the C-terminus and as the Q357R mutation does not alter putative phosphorylation sites, it is unlikely that the Q357R mutation affects the β-adrenergic regulation of the IKs current [31,32].

Thus, in vitro the Q357R mutation synergistically modified the gating properties and reduced the current density. However, the QTc interval of the affected patient was within the normal range. This is in agreement with recent findings that indicated that the IKs current forms a "repolarization reserve" [17]. In the absence of sympathetic stimulation, the contribution of the IKs current to the repolarization of the normal ventricular muscular action potential (AP) is rather limited. However, when the APD is prolonged by a reduction in IKr or in conditions of increased sympathetic stimulation, the IKs current limits action potential prolongation. The decrease of this "repolarization reserve" by the Q357R mutation would therefore cause an indirect impairment of ventricular repolarization. This reduction of the "repolarization reserve" could lead to a "silent" or "non-manifest" LQT1 phenotype, explaining the normal QTc interval of the patient.

In conclusion, the effect of the Q357R mutation on the gating properties of the channel combined with a reduced surface expression of the mutant protein should lead to a decrease in the native IKs current. Given the mild clinical manifestations, the Q357R mutation could be implicated in a "silent" LQT1 syndrome. This confirms the important role of the IKs channel in forming a "repolarization reserve".


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
We thank E. Mayeur and T. De Block for assistance in confocal microscopy. I.R. Boulet is a fellow with the "Fonds voor wetenschappelijk onderzoek Vlaanderen" (FWO Vlaanderen). This work was supported by grants from the "Fonds voor wetenschappelijk onderzoek Vlaanderen" (FWO-G.0085.04), and the Interuniversity Attraction Poles program P5/P19 of the Belgian Federal Science Policy Office.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 

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