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Cardiovascular Research 2005 67(3):476-486; doi:10.1016/j.cardiores.2005.04.036
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Copyright © 2005, European Society of Cardiology

Abnormal KCNQ1 trafficking influences disease pathogenesis in hereditary long QT syndromes (LQT1)

Andrew J. Wilsona, Kathryn V. Quinna, Fiona M. Gravesa, Maria Bitner-Glindziczb and Andrew Tinkera,*

aBHF Laboratories and Department of Medicine, University College London, 5 University Street, London, WC1E 6JJ, UK
bClinical and Molecular Genetics Unit, Institute of Child Health, 30 Guildford Street, London WC1N 1EH, UK

* Corresponding author. Email address: a.tinker{at}ucl.ac.uk

Received 8 December 2004; revised 20 April 2005; accepted 25 April 2005


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: In the hereditary long QT syndromes the commonest defect is in the K+ channel pore forming subunit, KCNQ1. In this study we investigated the role that abnormal KCNQ1 trafficking has in the pathogenesis of the hereditary long QT syndrome (LQT1).

Methods: We introduced nine missense and nonsense mutations occurring in LQT1 into the cDNA encoding KCNQ1 fused in frame to the green fluorescent protein. These mutations occur in syndromes that are inherited in both autosomal dominant and recessive fashions. We used biochemistry, electrophysiology and cell imaging to examine the behaviour of wildtype and mutant channel subunits expressed together with the auxiliary subunit KCNE1 expressed in CHO-K1 and C2C12 cells.

Results: We found that a number of mutations in KCNQ1 are retained in the endoplasmic reticulum and unable to translocate to the plasma membrane. Furthermore, some mutations act in a dominant negative fashion and have the ability to suppress the trafficking of wildtype channel. We use fluorescence resonance energy transfer microscopy to show that this occurs because of direct interaction between the mutant subunit and wildtype channel in the endoplasmic reticulum. Finally, a number of specific and nonspecific pharmacological tools are unable to promote the delivery of these mutants to the plasma membrane.

Conclusions: Our data revealed that channel trafficking may contribute to the pathogenesis of LQT1.

KEYWORDS K+ channels; Long QT syndrome; KCNQ1


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
An important cause of cardiac sudden death is the long QT syndrome (LQTS) [1] which is associated with a characteristic prolongation of the QT interval on the ECG. This predisposes the individual to torsade-de-pointes and subsequent sudden death due to ventricular fibrillation. There has been substantial progress in understanding the molecular basis of a number of rare hereditary syndromes. Mutations were identified in genes encoding ion channels namely the cardiac Na+ channel, two K+ channels (IKr and IKs) and their ancillary proteins [2–8]. IKr is thought to be a complex of the pore forming HERG subunit and perhaps (and more controversially) a modulatory subunit (MIRP1, KCNE2) whilst IKs is composed of the pore forming KCNQ1 and the auxiliary subunit KCNE1 [6,9,10]. Mutations in the {alpha} subunits of the K+ channels are by far the commonest cause of hereditary long QT syndromes with LQT1, due mutations in the protein KCNQ1, making up over 40% of cases. Two clinical syndromes are distinguished. In the Romano–Ward syndrome (RWS) inheritance occurs in an autosomal dominant pattern whilst in the rarer autosomal recessive Jervell–Lange–Nielsen syndrome (JLNS) there is profound hearing loss in addition to the prolonged QT interval and predisposition to sudden death [11–14].

A substantial body of work has investigated the electrophysiological consequences of ion channel mutations in these syndromes. In RWS due to KCNQ1 mutations a dominant negative mechanism is thought to act with a mutant subunit able to abrogate the function of the tetramer [15,16]. In JLNS, KCNQ1 mutations generally lead to a loss of function without pronounced dominant negative effects [17,18]. However, there is a considerable spectrum of functional effects in each disease and there is evidence for variable penetrance of some mutations [18,19]. We have previously studied the functional effects of a wide spectrum of KCNQ1 mutations in JLNS [18]. In particular, we wanted to know why heterozygotic carriers did not generally manifest symptoms. Our in vitro observations broadly tallied with the above hypothesis for the mechanism of the disease. Thus mutations in KCNQ1 were nonfunctional when coexpressed with KCNE1 and generally did not lead to a dominant negative effect when coexpressed at a 1:1 expression level with wild-type channel. However, we did find exceptions, for example, the mutation E261D had a profound dominant negative effect on wildtype currents at this expression ratio [18]. In addition, a mutation (L273F) found in RWS did not lead to a large dominant negative effect.

It has generally been assumed that the mutant subunits (five in total in the study cited) are in non-functional complexes at the plasma membrane [20]. More recently three specific mutations in KCNQ1 (T587M, DeltaS276, Ala178fs/105) have been reported to have problems in membrane delivery and variable effects on the trafficking of the wildtype subunit on coexpression [21–23]. Nevertheless it is clear in a number of other hereditary diseases that defective trafficking is a major component in the pathogenesis of the clinical syndrome. The classic example is cystic fibrosis where the commonest mutation, {Delta}F1388 in the cystic fibrosis transmembrane conductance regulator, is retained intracellularly at 37 °C. When the cells are cooled a functional chloride channel is expressed at the plasma membrane [24,25]. It is not clear whether trafficking considerations are generally important in the long QT syndrome associated with mutations in KCNQ1 (LQT1). It is this question we address here.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Molecular biology
A fusion between human KCNQ1 and GFP was created using PCR. The long isoform of KCNQ1 minus the stop codon (a gift from Dr. Barhanin) was amplified by PCR using a high fidelity polymerase (Vent DNA polymerase, New England Biolabs) on EcoRI/XbaI ends into pcDNA3. Enhanced GFP was amplified by PCR and cloned in frame to the C-terminus of KCNQ1 on XbaI/ApaI ends. A similar strategy was used for the mutants. Mutations were introduced into wild-type KCNQ1 using either site-directed mutagenesis or splicing-by-overlap extension (SOE) PCR. Mutations were confirmed by DNA sequencing. Cyan fluorescent protein (eCFP, Clontech) and yellow fluorescent protein (eYFP, Clontech) constructs were generated by subcloning from the GFP tagged constructs. KCNE1 was cloned into pcDNA3.1/Zeo on BamHI/EcoRI ends.

2.2. Western blotting
Western blotting was undertaken as previously described [26]. We used a custom generated rabbit polyclonal antibody raised to the N-terminus of KCNQ1 (sequence of ETRGSRLTGGQGRVYNFLERC). The peptide was linked to KLH, the antisera raised in rabbits handled according to UK Home Office regulations and NIH guidelines (Regal Group Ltd, UK) and affinity purified as in our previous studies [27].

2.3. Cell culture
CHO-K1 cells and C2C12 cells (obtained from the ATCC) were cultured in Ham F12 and MEM media, respectively, with 10% fetal calf serum and transiently transfected with 500 ng of cDNA using Lipofectamine Plus according to manufacturers' instructions (Invitrogen). Cells were allowed to grow for 24 h before being seeded onto glass coverslips.

2.4. Electrophysiology
KCNQ1 currents were recorded from transfected CHO-K1 cells using the whole-cell configuration of the patch-clamp technique. Transfected cell was identified by epifluorescence. Cells were dialysed with a solution containing in mM: 150 KCl, 5 EGTA, 10 HEPES, 2 MgCl2, 1 CaCl2 and 5 (Na)2ATP (pH 7.2). Bath solution contained in mM: 150 NaCl, 5 KCl, 10 HEPES, 2 MgCl2 and 1 CaCl2 (pH 7.4). Currents were recorded by holding the cell at a voltage of –80 mV followed by stepped depolarisations from –80 to +80 mV for 6 s in 10 mV increments, followed by a repolarising pulse to –20 mV for 3 s (to measure tail currents) and back to –80 mV. Currents are normalised to cell capacitance (current density, pA/pF). Data were acquired using an Axopatch 200B amplifier (Axon Instruments, USA) and analysed using Clampfit and Microcal Origin software. Series resistance was at least 70% compensated using the amplifier circuitry. Pipette resistance is ~1.5–2.5 M{Omega} for whole-cell recording and pipette capacitance was reduced by coating the tip with a parafilm-oil suspension. Steady-state activation, activation and deactivation kinetics were measured as previously described [18].

2.5. Microscopy
Live cells were imaged using a Biorad Radiance 2100 laser scanning confocal Nikon TE300 microscope (Biorad, UK). EGFP was excited using an argon 488 nm laser and emission recorded using a HQ515/30 filter. DsRed2 was excited using a helium/neon laser at 543 nm and images collected with an HQ590/15 filter set. Colocalisation analysis was performed using Laserpix software (Biorad, UK). CFP was excited with a 457 nm laser line and images were obtained using a 470–500 nm band pass filter. YFP was excited with a 514 nm laser line and emission measured between 530 and 570 nm. The FRET channel was excited using a 457 nm laser line and emission measured between 530 and 570 nm. All images were acquired sequentially. Intensities in each of the three channels were determined from membrane-delimited regions of interest drawn by hand using the LaserPix software. In all the figures the bar indicates 10 µm.

For ER localisation, the colocalisation coefficient is the sum of all the green pixel intensities (GFP) which have a red component (ER) divided by the sum of all the green pixel intensities in the image. Further information is available at http://microscopy.biorad.com/reference/technical/TECH11.PDF. Such analysis proved to be robust however it is not without subjective elements including the initial optimisation of imaging conditions and subsequent thresholding during analysis.

The background-subtracted intensities were analysed to determine FRET ratios using three cube protocols. Sixteen-bit images were obtained with identical laser powers, photomultiplier gain and pinhole size. FRET ratios were calculated as previously described [28,29]. Images were pseudocoloured, filtered and converted to 24 bit RGB files for display (TIFF or JPEG). Chromanol293B and HMR1558 were a kind gift of Aventis pharmaceuticals.

2.6. Statistical analysis
Data were compared using either unpaired t-test or one-way ANOVA with Bonferroni post-hoc test for multiple comparisons. Data were considered to be statistically significant when P<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. A review of the mutations used in this study
Here we study the trafficking of our previously described mutants that occur in JLNS (R243H, E261D, R518X, Q530X, 1008delC and R594Q) [18,30]. X refers to the generation of a stop codon at the residue stated and 1008delC causes a frameshift at codon 337 leading to a premature stop codon at position 353. We also study some mutations described in RWS namely E261K, L273F and R366Q [18,31–33]. The clinical details are given in the references above and in particular Table 1 in Ref. [18].

3.2. Characterisation of KCNQ1 fused to GFP
To investigate the role of trafficking in LQT1 we fused KCNQ1 at the C-terminus in-frame to eGFP. We first established that this protein was functional and behaved in a manner analogous to wild-type untagged protein. CHO-K1 cells were transiently transfected with KCNQ1-GFP+KCNE1 and compared to those transfected with KCNQ1+KCNE1. The resulting currents were recorded using the whole-cell configuration of the patch-clamp technique. A large voltage-dependent current with a slow activation time course, typical of cardiac IKs current was present in both sets of cells (Fig. 1A). There was a trend for higher expression with KCNQ1-GFP+KCNE1 though this was not statistically significant. We also measured the kinetics of activation (Fig. 1B) and the steady-state activation from the tail currents (Fig. 1C). Neither of these were significantly different between KCNQ1-GFP+KCNE1 and KCNQ1+KCNE1. Finally deactivation kinetics were measured at –40 mV (after a +80 mV depolarising pulse). The mean tau for deactivation was 1057 ± 143 ms (n = 5) for KCNQ1+KCNE1 and 785 ± 114 ms (n = 10) for KCNQ1-GFP+KCNE1. The reciprocal of time is normally distributed and comparison after this transformation showed the difference not to be significant (NS, P = 0.16). Thus KCNQ1-GFP is functional and behaves in a similar manner to wild-type untagged protein.


Figure 1
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  		Fig. 1 Effects of GFP tagging on KCNQ1 channel function. (A) Representative whole cell recordings and mean current voltage relationships. Mean activation time constants (B) and steady-state activation as determined from normalised tail current amplitude. The latter data points are fitted with two Boltzmann functions using non-linear regression: V0.5=14.4 ± 1 mV and slope factor 12.7 ± 0.9 mV for KCNQ1-GFP+KCNE1 and V0.5=15.9 ± 2.3 mV and slope factor 17.3 ± 2.1 mV for KCNQ1+KCNE1. KCNQ1-GFP+KCNE1 (n = 10, lhblk) and KCNQ1+KCNE1 (n = 5, {blacktriangleup}). Data are shown as mean ± s.e.m. (D) A Western blot showing expression of the indicated mutants. Molecular weight markers in kDa as indicated. *marks a background band and KCNQ1 the expected mobility of full-length KCNQ1.

 
Known LQTS mutations were incorporated into KCNQ1-GFP and the resulting constructs transfected into CHO-K1 cells. After 48 h, cells were lysed, and diluted with SDS loading buffer and fractionated by SDS-PAGE. The molecular weight of these chimaeric proteins was assessed by Western blotting using a custom-made rabbit polyclonal antibody directed against a unique sequence in the N-terminus of KCNQ1. Mutants expressed protein of the correct approximate molecular weight (Fig. 1D). The apparent variation in protein expression is related to variable transfection efficiencies as all the mutants expressed robustly in single-cells as judged by epifluorescence (see below).

3.3. ER retention of KCNQ1 and disease causing mutants
Misfolded proteins are recognised by a quality control mechanism that leads to the ER retention (and subsequent degradation) of the protein [34]. Thus we sought to establish a quantitative microscopy assay using a laser scanning confocal microscope to measure the degree of ER retention. CHO-K1 cells were transfected with KCNQ1-GFP with and without KCNE1 expression and a DsRed2 protein that has been engineered to be retained in and mark the ER. It is apparent in Fig. 2A that both KCNQ1-GFP and that KCNQ1-GFP coexpressed with KCNE1 are largely present on the plasma membrane of the cell. This was confirmed when we quantitated the level of colocalisation of KCNQ1-GFP with DsRed2-ER, i.e., the percentage of green pixels that are also red (Fig. 2B). It was also apparent that coexpression of KCNE1 subtly but significantly enhanced the exit of KCNQ1-GFP from the ER.


Figure 2
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  		Fig. 2 Effects of KCNE1 on the trafficking of KCNQ1-GFP. (A) Representative confocal images of CHO-K1 cells transiently transfected with KCNQ1-GFP (top panels) and together with KCNE1 (bottom panels). Images are shown for GFP alone, DsRed2-ER, and the merged image. Co-localisation between KCNQ1-GFP and DsRed2-ER appears as yellow. (B) Mean data showing the proportion of KCNQ1-GFP: ER co-localisation in the absence and presence of KCNE1. Data are shown as mean ± s.e.m. (n = 44–102 cells, *P<0.05 compared to control).

 
We next examined the behaviour of a series KCNQ1 mutants that occur in both RWS and JLNS (see above and Fig. 3A and B). Transfection of GFP tagged KCNQ1 mutants together with KCNE1 in CHO-K1 cells revealed a spectrum of behaviour. However there was significant retention of a large number of the mutants in the ER compared to KCNQ1-GFP with KCNE1 alone. Quantitation revealed a range of values for ER retention (Fig. 3C). However both RWS and JLNS mutants were retained and even relatively conservative point mutations (e.g. E261D) can be profoundly retained. Is this trafficking behaviour a unique feature of expression in CHO-K1 cells? We addressed this issue and expressed the wildtype channel and two representative mutants (E261D, R366Q) in a murine myocyte cell line (C2C12). We undertook a similar analysis to that shown in Fig. 3 and observed a similar pattern of subcellular distribution both qualitatively and quantitatively. ER retention, as assayed by colocalisation with ER-DsRed, was 0.27 ± 0.03 (n = 18) for KCNQ1-GFP+vKCNE1, 0.63 ± 0.02 (n = 12) for E261D-GFP+KCNE1 and 0.36 ± 0.02 (n = 11) for R366Q-GFP+KCNE1.


Figure 3
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  		Fig. 3 Effects of LQT-causing mutations on the trafficking of KCNQ1-GFP. (A) Representative confocal images of mutant KCNQ1-GFP proteins. Images are shown for GFP alone, DsRed2-ER, and the merged image. Co-localisation between KCNQ1-GFP and DsRed2-ER appears as yellow. All mutants are cotransfected with KCNE1. (B) A cartoon showing the distribution of mutations. (C) Mean data showing the proportion of ER co-localisation for 9 different KCNQ1 mutations (+KCNE1) compared to control (KCNQ1-GFP+KCNE1). n = 15–55, *P<0.05, #P<0.001.

 
3.4. Dominant negative effects of trafficking mutants
Do the trafficking defective mutants have a dominant effect over the wildtype channel? We cotransfected KCNQ1-GFP together with the untagged mutants E261D and 1008delC. At a 1:1 expression ratio of KCNQ1-GFP to mutant, E261D led to a pronounced retention of the wildtype channel in the ER. In contrast 1008delC, R518X and Q530X did not lead to retention at a 1:1 ratio but 1008delC and Q530X did at a 1:3 ratio (Fig. 4). We complemented these studies by examining the effect that these mutants had on whole-cell currents. Coexpression of E261D-GFP and E261K-GFP with KCNQ1-GFP/KCNE1 at a 1:1 ratio led to a decrease in tail current density (Fig. 5) compatible with a dominant negative effect and consistent with the effect on trafficking (Fig. 4 and see below).


Figure 4
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  		Fig. 4 Dominant-negative effects of some mutant KCNQ1 proteins. CHO-K1 cells were transfected with KCNQ1-GFP+KCNE1 together with an untagged mutant protein. (A) Representative confocal images showing some LQT1 mutants expressed at either a 1:1 ratio (WT KCNQ1-GFP: mutant KCNQ1 untagged) or at a 1:3 ratio. Data recorded from these cells are presented in (B). Data are mean ± s.e.m. from 18–30 cells. #P<0.001.

 

Figure 5
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  		Fig. 5 Patch-clamp studies of dominant-negative mutants. (A) Representative traces of KCNQ1-GFP with KCNE1 (left) and E261K KCNQ1-GFP plus KCNQ1-GFP with KCNE1 (right) recorded from CHO-K1 cells normalised to cell capacitance. (B) Mean peak tail currents at –20 mV following depolarisation to 80 mV for KCNQ1-GFP plus KCNE1, E261K KCNQ1-GFP plus KCNQ1-GFP (1:1) and KCNE1, and E261D KCNQ-GFP plus KCNQ1-GFP (1:1) and KCNE1. Data are shown as mean ± s.e.m. *P<0.05 compared to control.

 
3.5. FRET microscopy reveals interaction between KCNQ1 and disease causing mutants
We further investigated the mechanism for this effect in living cells using fluorescence resonance energy transfer (FRET) microscopy. It is increasingly used in biological settings to report the physical interaction of proteins in living cells [29,35,36]. We sought to establish if FRET could be reported between KCNQ1 with CFP fused at the C-terminus (KCNQ1-CFP) and KCNQ1 with YFP fused at the C-terminus (KCNQ1-YFP). Using laser scanning confocal microscopy, we examined whether there was basal FRET between these two proteins transiently expressed in CHO-K1 cells. We collected three images corresponding to a CFP, FRET and YFP channel in cells expressing each construct alone or the two together (Fig. 6). After transfection of each individual species, it is possible to obtain parameters that quantify bleed through into the various channels (three-cube correction) [28,29] and calculate a net FRET image and a FRET ratio on a defined region of interest (see Methods). We first characterised the system by coexpressing the D2S dopamine receptor tagged with CFP with the M4 muscarinic receptor tagged with YFP and also D2-CFP with KCNQ1-YFP. D2-CFP and M4-YFP are membrane localised G-protein coupled receptors that are not thought to form heterodimers [37]. In addition, there is no precedent for the interaction of D2S with KCNQ1. Thus neither of these pairs of intrinsic membrane protein would be expected to interact and thus can be used as negative controls. This was indeed the case as there was little evidence of net FRET after image processing and the FRET ratios calculated at the membrane were ~1 (Fig. 6). In contrast, image processing revealed a potential FRET signal located at the plasma membrane with the coexpression of KCNQ1-CFP with KCNQ1-YFP (and KCNE1). Furthermore analysis of membrane delimited regions of interest led to a calculated FRET ratio of ~1.5 (Fig. 6B). Thus it seems we can report the physical interaction of KCNQ1 subunits in the tetramer. All constructs studied expressed to similar levels.


Figure 6
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  		Fig. 6 FRET studies on interactions between mutant and wild-type KCNQ1 proteins. Panel A shows CHO-K1 cells expressing the CFP and YFP tagged proteins (+KCNE1 except D2-CFP/M4-YFP), together with their respective FRET images. 3-cube FRET ratios were calculated as according to the methods and representative images are shown. Pixel intensity for each column is indicated by the colour bar at the top. (B) Graph indicating mean ± s.e.m. FRET ratio under the respective conditions (+KCNE1 except D2-CFP/M4-YFP), **P<0.01 and #P<0.001 when compared to control (D2-CFP+KCNQ1-YFP+KCNE1), n = 19–31.

 
We next used this assay to report the interaction of selected mutants (E261D-YFP, E261K-YFP, 1008delC-YFP, R518X-YFP and Q530X-YFP) with KCNQ1-CFP expressed at a 1:1 ratio. Image analysis revealed intracellular FRET signal from the ER between KCNQ1-CFP and E261K-YFP and E261D-YFP but not between KCNQ1-CFP and 1008delC-YFP, R518X-YFP and Q530X-YFP. Thus this assay reveals that the dominant negative effect of E261K and E261D arises because of the interaction between the wildtype channel and the E261 mutants. In contrast, the wildtype channel is able to escape the ER when coexpressed at a 1:1 expression ratio with 1008delC, R518X and Q530X mutants as it does not significantly interact with the wildtype channel. The 1008delC mutant does not contain the putative assembly domain in the C-terminus whereas the E261 mutants do [17].

3.6. Pharmacological and other interventions fail to relieve ER retention
There is a precedent for the modulation of trafficking of mutant membrane proteins by temperature and pharmacological agents [24,25,38,39]. We examined the effects of temperature (Fig. 7A) and the pharmacological agents diisothiocyanostilbene disulfonic acid (DIDS), mefenamic acid, chromanol 293B and HMR1558 (Fig. 7B and C). DIDS and mefenamic acid have been reported to activate KCNQ1/KCNE1 currents and rescue LQT5 mutants [40,41]. Chromanol 293B and HMR1556 are specific inhibitors of the KCNQ1/KCNE1 channel complex [42,43]. None of these agents were effective in reducing ER retention and plasma membrane delivery. We also tried the proteasomal inhibitor MG132 with a representative mutant and this was ineffective (not shown). Glycerol, a non-specific chaperone, led to pronounced cell death in our hands (not shown).


Figure 7
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  		Fig. 7 Effects of temperature and channel modulators on trafficking. (A). Mean proportion of ER co-localisation recorded from cells at either 27 or 37 °C after 24 h incubation for each of the mutants listed. KCNE1 was cotransfected with all mutants. KCNQ1-GFP is not transfected with KCNE1 and the dotted line indicates the expected degree of retention of KCNQ1-GFP+KCNE1. (B) The effects of the IKs current activators, DIDS (100 µM) and mefenamic acid (100 µM), on the ER co-localisation of 3 KCNQ1 mutants (E261D, E261K and 1008delC) are shown. All conditions include cotransfection with KCNE1. (C) The effects of the IKs current inhibitors, chromanol 293B (10 µM) and HMR-1556 (100 nM) on the trafficking of WT, R243H and E261D KCNQ1-GFP are shown. All data are expressed as mean ± s.e.m, n = 14–19. All conditions include cotransfection with KCNE1.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The importance of trafficking in influencing the pathogenesis of the long QT syndrome has been little appreciated in LQT1. There are some recent studies which demonstrate failure of cell surface expression of three mutant channels potentially leading to haploinsufficiency (T587M and DeltaS276) or a dominant negative effect (A178fs/105) [21,22,44]. The major novel findings in this study are that a number of mutations in KCNQ1 in both RWS and JLNS are retained in the ER and retention can be dominant over the wildtype subunit due to direct protein–protein interaction in the ER. Indeed the majority of KCNQ1 mutations we have studied in LQT1 show a degree of such behaviour and they include both nonsense and missense mutations. Even very conservative mutations, such as E261D, can display a profound defect in channel biogenesis. One advantage of the assay we use is that it reveals relative as well as absolute defects and this may account for the failure of other investigators to see subtle but significant abnormalities [20]. Alternatively, mutations studied by other laboratories may behave more like L273F studied here. A number of these mutations associated with the dominantly inherited RWS lead to a suppression of wildtype channel trafficking. Thus it is clear that there is substantial potential for the phenotype in LQT1 in both RWS and JLNS to be determined by failure of channels to be delivered through the secretory pathway and not simply the presence of abnormally functioning channels at the surface of the cell. The data is also consistent with our previous observations that the E261D mutation can exert a profound dominant negative effect on current amplitude when coinjected with wild-type channel in Xenopus laevis oocytes [18]. We have confirmed this observation in mammalian cells with fluorescently tagged mutants in this study. We have also shown that E261K leads to a similar dominant negative suppression of current as might be expected given the RWS phenotype. Other investigators were unable to see such effects though there was no control for protein expression [32]. Indeed the trafficking phenotype matches the clinical phenotype too. For example, 1008delC, R518X and Q530X occur in JLNS with largely asymptomatic heterozygotes and the mutation does not lead to a prominent dominant negative effect [18]. We find that this mutation does not exit the ER and it is largely unable to interact with wildtype channel subunits at a 1:1 expression ratio. In contrast, E261K occurs in RWS, is ER retained, can interact with wildtype subunits and as a result lead to a dominant negative effect on channel trafficking (and current). Our results with FRET microscopy and dominant negative suppression of wildtype trafficking are consistent with the C-terminal assembly domain [17]. However we have also argued that other determinants, in the N-terminus and transmembrane domains, may play a role at higher mutant concentrations [18]. Indeed 1008delC and other mutations lacking the assembly domain can suppress trafficking at a 1:3 expression ratio. These results too are consistent with our observations here and in previous electrophysiological studies of these mutants [18].

The role of defective trafficking is better appreciated in LQT2 where a number of mutations in HERG are known to lead to ER retention [45]. Furthermore it is possible to "rescue" these mutations by treating the cells with pharmacological blockers of the channel. We tried a number of approaches with our mutations in KCNQ1 including treatment with specific pharmacological agents known to act on KCNE1 (DIDS and mefenamic acid) and the pore forming subunit (chromanol 293B and HMR1556). These agents did little to improve the ER retention of a selection of the mutants. There are other more non-specific methods for enhancing trafficking and one of the most general is temperature [24,25]. However cooling did not improve the delivery of the mutant KCNQ1/KCNE1 complex to the plasma membrane. Thus to date our efforts at potentially rescuing these mutations has not been successful.

There has been considerable recent interest in the role of small peptide motifs in determining the subcellular distribution of potassium and other channels [46–48]. However the mutations we have studied are distributed throughout the protein and range from very conservative point mutations to frame shift nonsense mutations. It is thus likely that these do not specifically disrupt a particular trafficking process but rather are trafficking deficient because of a general cellular response and quality control mechanism that recognises aberrant mutant proteins [49]. A recent study has identified a small region (amino acids 610–620) that is important for cell surface expression [44]. Our data show that mutations outside this domain can be trafficking deficient to varying degrees. Indeed residues in the S4–S5 linker may be another "hot spot" as E261D and E261K are ER retained. Another novel aspect of our data is that KCNE1 enhances the cellular redistribution of KCNQ1 to the plasma membrane. The effect is modest, and this may account for the failure of other investigators to see it [50], but it will contribute with the increase in single-channel conductance [51,52] to the enhancement in the magnitude of whole-cell currents seen after co-expression of KCNQ1 with KCNE1.

Finally, in this study we use a FRET based microscopy assay to examine the potential interaction of wildtype and mutant subunits directly in the living cell. We combine this with the preservation of information about subcellular location to directly demonstrate that the dominant negative effect of some mutants on the trafficking of the wild-type channel is due interaction between the two subunits in the ER. In addition, in principle we may be able to investigate conformational changes [36] occurring with membrane depolarisation and we are actively pursuing such studies.


   Acknowledgements
 
This work was supported by the British Heart Foundation.


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

  1. Schwartz P.J., Periti M., Malliani A. The long QT syndrome. Am Heart J (1975) 89:378–390.[CrossRef][ISI][Medline]
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