Cardiovascular Research

Cardiovascular Research 2005 67(3):520-528; doi:10.1016/j.cardiores.2005.03.007

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Copyright © 2005, European Society of Cardiology

Atrial fibrillation-associated minK38G/S polymorphism modulates delayed rectifier current and membrane localization

Joachim R. Ehrlich^a,1, Stephen Zicha^a,c,1, Pierre Coutu^a, Terence E. Hébert^b,c and Stanley Nattel^a,c,*

^aDepartment of Medicine and Research Center, Montreal Heart Institute and University of Montreal, 5000 Belanger Street East, Montreal, Quebec, Canada, H1T 1C8
^bDepartment of Anesthesiology, Montreal Heart Institute and University of Montreal, Montreal, Quebec, Canada
^cDepartment of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

^* Corresponding author. University of Montreal, Montreal Heart Institute, Department of Medicine, 5000 Belanger Street East, Montreal, Quebec, Canada H1T 1C8. Tel.: +1 514 376 3330; fax: +1 514 376 1355. Email address: stanley.nattel{at}icm-mhi.org

Received 22 December 2004; revised 16 February 2005; accepted 10 March 2005

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
Background: Atrial fibrillation (AF) is a common acquired arrhythmia with multi-factorial pathogenesis. Recently, a single nucleotide polymorphism (SNP, A/G) at position 112 in the KCNE1 gene, resulting in a glycine/serine amino acid substitution at position 38 of the minK peptide, was associated with AF occurrence (AF more frequent with minK38G); however, the functional effect of this SNP is unknown.

Methods and Results: We used patch clamp recording, confocal microscopy and protein biochemistry to study the effect of this SNP on delayed-rectifier current expression and mathematical simulation to identify potential functional consequences. The density of slow delayed rectifier current (I_Ks) resulting from co-expression with KvLQT1 was smaller with minK38G (e.g. at +10 mV: 50 ± 7 pA/pF in Chinese hamster ovary (CHO) cells, 45 ± 14 pA/pF for COS-7 cells) compared to minK38S (93 ± 17 pA/pF, 104 ± 23 pA/pF, respectively, P<0.05 for each). I_Ks kinetics and voltage-dependence were unaffected. Currents resulting from co-expression of human ether-a-go-go-related gene (HERG) were similar for minK38G and minK38S, e.g. upon repolarization from +10 to –50 mV: tail currents 23 ± 4 pA/pF versus 22 ± 5 pA/pF (P = ns). KvLQT1 membrane immunofluorescence was less in CHO cells co-expressing minK38G versus minK38S, and surface expression of KvLQT1, as determined by labelling with streptavidin/biotin, was increased with minK38S co-expression. Computer simulations with a human atrial action potential model predicted that the minK38G SNP would slightly prolong the atrial action potential and reduce the frequency for alternans behaviour. In the presence of reduced repolarization reserve, these effects were enhanced and under specific conditions early afterdepolarizations occurred.

Conclusions: The minK38G isoform is associated with reduced I_Ks, likely due to decreased KvLQT1 membrane expression. This study reveals a novel amino acid determinant of the minK-KvLQT1 interaction, and if the role of minK38G in AF is confirmed, would suggest mechanistic heterogeneity in genetic determinants of AF.

KEYWORDS Arrhythmia (mechanisms); Gene polymorphisms; Ion channels; K-channel; Repolarization

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
Atrial fibrillation (AF) is a common arrhythmia with a heritable component [1,2]. The presence of a genetic susceptibility to AF development is supported by the finding that children of subjects enrolled in the Framingham heart study were at increased risk for later AF if their parents were also affected by the arrhythmia [3]. Autosomal dominant familial AF has been described and the disease has been linked to the locus 10q22–q24, but the precise gene defect in these families remains unknown [4]. A variety of genetic abnormalities predispose to AF along with other arrhythmic and/or cardiomyopathic syndromes. These include lamin A/C mutations [5,6], angiotensin gene polymorphisms [7], the Brugada syndrome [8], the short QT syndrome [9], Long QT Syndrome (LQTS) type 4 with ankyrin mutation [10,11], and possibly other forms of congenital LQTS [12]. The only known forms of monogenic familial AF occurring in the absence of other recognized cardiac abnormalities are caused by gain-of-function mutations in KCNQ1 and KCNE2 genes (which encode KvLQT1 and minK-related peptide 1, MiRP1, K⁺-channel subunits respectively) resulting in either increased I_Ks with altered current characteristics or increased potassium background current [13,14]. Taken together, these data suggest that various–possibly subtle–changes in the atrial environment may lead to a predisposition to an increased AF propensity. These alterations comprise distinct structural and electrophysiological mechanisms that lead to a final common vulnerable substrate for AF.

Recently, a single-nucleotide-polymorphism (SNP) in the KCNE1 gene (A to G at position 112), leading to a glycine substitution for serine at amino-acid position 38, has been reported to be associated with increased AF incidence [15,16]. The odds ratio (OR) for AF with one minK38G allele was 2.16 and increased to 3.58 with 2 minK38G alleles (95% confidence-interval 1.38–9.27) [16]. The functional consequences of this polymorphism remained unclear. The present study examined the hypothesis that a glycine at position 38 of the minK protein alters slow (I_Ks) and/or rapid (I_Kr) delayed-rectifier current that results from co-expression with the corresponding -subunits KvLQT1 and HERG, respectively.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
2.1. Polymerase chain reaction (PCR)
The original minK clone (GenBank accession number NM000219) contained glycine at position 38, so minK38S was created by overlap extension PCR, with the primers shown in Table 1. The construct was flanked by HindIII (5') and BamHI (3') restriction sites and subsequently re-ligated into pcDNA3.1. PCR products were verified by cDNA sequencing, which was confirmed on two separate occasions for each construct. MinK, KvLQT1 (GenBank accession number NM000218) and HERG (NM000238) cDNA were all subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA, USA).

View this table: [in this window] [in a new window]   Table 1 Primers for minK mutagenesis

 
2.2. Cell culture
Chinese hamster ovary (CHO) and Cercopithecus aethiops kidney (COS-7) cells (ATCC, Manassas, VA) were cultured at 37 °C with 5% CO₂ in F12 or DMEM medium (Invitrogen Canada, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated fetal bovine serum along with 100 U/mL penicillin and 100 µg/mL streptomycin. Transient transfections were performed with Lipofectamine-plus (Invitrogen Canada, Burlington, Ontario, Canada) and 0.5-µg minK cDNA. Co-transfection was performed with KvLQT1 (1 µg cDNA) or HERG (0.5 µg cDNA). Cells were studied with the use of whole-cell patch clamp recording or immunofluorescence confocal microscopy 36–48 h after transfection. Co-transfected green fluorescent protein (GFP; 0.1 µg cDNA) served as a transfection marker. Twenty-five centimeter square flasks (Sarstedt Inc., Montreal, Quebec, Canada) of CHO cells were transfected for Biotin–Streptavidin assays.

2.3. Confocal microscopy
For immunofluorescent studies, transiently transfected CHO cells were plated on sterile glass coverslips for 24 h. Cells were fixed (20 min) with 2% paraformaldehyde (Sigma-Aldrich, Oakville, Ontario, Canada) and washed 3 times (5 min each) with phosphate buffered saline (PBS). After blocking with 5% normal donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) and 5% bovine serum albumin (BSA, Sigma) cells were permeabilized with 0.2% Triton X-100 (Sigma) for 1 h. They were then incubated overnight at 4 °C with primary antibodies: goat anti-minK (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-KvLQT1 (Chemicon, Temecula, CA, USA) at 1:200 dilution, followed by three 5-min washes with PBS and a 1 h incubation with secondary antibodies (anti-goat IgG labelled with fluorescent isothiocyanate [FITC] for minK and anti-rabbit IgG with tetra-methyl-rhodamine-isothiocyanate [TRITC] for KvLQT1). Confocal microscopy was performed with a Zeiss LSM-510 system. TRITC (red) and FITC (green) were excited at 543 and 488 nm with HeNe- and Ar-Lasers respectively, emitting fluorescence at 566 and 525 nm.

Confocal microscopy experiments were performed on the same day for both groups, with identical parameters used for all manipulations. Control experiments omitting primary antibodies and with non-transfected CHO cells revealed absent or very low-level background staining.

2.4. Electrophysiology
Currents were recorded in the whole-cell patch-clamp configuration at 36 ± 0.5 °C with an Axopatch 200B amplifier and pClamp software (V6.0, Axon Instruments, Foster City, CA, USA). Borosilicate glass electrodes had 2–3 M tip resistances when filled. Mean compensated cell-capacitances were 15.0 ± 1.1 pF for transfected CHO and 19.6 ± 1.9 for transfected COS-7 cells, with no significant differences between cells transfected with minK38G or 38S. Liquid junction potentials averaged 7.5 ± 0.5 mV and were not corrected.

The extracellular solution for current recording contained (mmol/L) NaCl 136, KCl 5.4, MgCl₂ 1, CaCl₂ 1, NaH₂PO₄ 0.33, HEPES 5 and dextrose 10 (pH 7.35 with NaOH). Internal solution contained (mmol/L) K-aspartate 110, KCl 20, MgCl₂ 1, MgATP 5, GTP (lithium salt) 0.1, HEPES 10, Na-phosphocreatine 5 and EGTA 5.0 (pH 7.3 with KOH).

Currents were elicited with 2-s depolarizing pulses from a holding potential (HP) of –80 mV and an inter-pulse interval of 12 s, with tail currents observed during 2 s repolarizations to –50 mV for I_Ks (4 s for I_HERG). Clampfit (Axon Instruments) and GraphPad Prism V3.0 were used for data analysis.

2.5. Biotin–Streptavidin surface labelling and immunoblotting
CHO cells were transfected with KvLQT1 and minK cDNA of either minK 38G or 38S as indicated above in 25 cm² flasks (Sarstedt Inc., Montreal, Quebec, Canada), and kept for 24 h in a similar fashion to cells used for patch-clamp experiments. Cells were washed three times with ice-cold PBS (pH 8.0) to remove amine-containing culture media from the cells. Sulfo-NHS-LC-Biotin reagent (10 mg, Pierce Ltd, Rockford, IL, USA) was added to 25 cm² cell-containing flasks and the mixture was allowed to incubate at room temperature for 30 min. Cells were then washed three times with PBS-containing glycine (100 mmol/L) to quench excess biotin. The cells were then scraped from the flask and resuspended in a lysis buffer containing 5 mmol/L Tris–HCl (pH 7.4), 2 mmol/L EDTA, 10 mg/mL benzamidine, 5 mg/mL leupeptin, 5 mg/mL soybean trypsin inhibitor, and 1% Triton X-100 and allowed to lyse for 45 min with gentle rotation. Lysates were then centrifuged for 15 min at 500 x g at 4 °C and the supernatant was conserved and centrifuged at 45,000 x g for 15 min at 4 °C. The resulting pellet was re-suspended in PBS containing 0.1% sodium-dodecyl-sulfate (SDS). The protein suspension was incubated along with streptavidin immobilized on beads for 1 h at room temperature. Any proteins not linked to biotin (and therefore not expressed at the cell surface) were removed by four washes with PBS and 0.1% SDS. The samples were suspended in an appropriate volume of Laemmli buffer and 75 mmol/L Tris–HCl, 12.5 mmol/L MgCl₂ and 5 mmol/L EDTA and then boiled to elute the protein before loading for SDS-PAGE electrophoresis. Streptavidin precipitates were analyzed on 7.5% SDS-PAGE gels. Proteins were transferred to PVDF membranes and blotted with anti-KvLQT1 antibody (Chemicon, 1:400). Bands were visualized with chemiluminescence imaging (Western Lightning, Perkin-Elmer Life Sciences, Torrance, CA, USA).

2.6. Mathematical simulation of consequences of SNP effects on I_Ks
Computer simulations were performed to study the effect on human atrial action potential duration (APD) of changes in I_Ks density of the order produced by the minK SNPs studied, with the use of a mathematical model of the human atrial action potential. The Courtemanche–Ramirez–Nattel model [18] was used, and several maximal conductance parameters (g_Ks, g_Kr, g_K1, and g_Ca,L) were allowed to vary from the original model as described in the Results Section. Numerical integration was performed using the 4th order with an adaptive time-step Runge–Kutta–Merson algorithm [17]. The results were obtained for the 100th action potential (AP) when stimulated from rest at specific pacing frequencies.

2.7. Data analysis
Data are presented as mean ± S.E.M. A two-tailed P<0.05 (Student's t-test) was considered statistically significant.

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
3.1. Electrophysiological studies–KvLQT1 co-expression
Fig. 1A and B show representative currents from CHO cells expressing KvLQT1 with minK38G and minK38S, respectively. Although the form of the currents appeared similar, currents tended to be larger with minK38S co-expression. Overall, I_Ks densities were smaller for minK38G-expressing cells (Fig. 1C); e.g. at +10 mV, 50.1 ± 6.9 pA/pF for minK38G vs. 92.5 ± 17.4 pA/pF for minK38S (n = 20 cells each, P<0.05). Tail currents were also smaller with minK38G (following a pulse to +10 mV, 11.1 ± 1.8 pA/pF versus 19.2 ± 2.9 pA/pF for minK38S, P<0.05). Half-activation voltages (V₅₀) obtained from Boltzmann fits to normalized tail currents did not differ: 11.6 ± 3.5 mV for minK38G vs. 11.4 ± 1.9 mV for minK38S (P = ns, Fig. 1D). Activation time constants were similar (Fig. 1E); e.g. at +10 mV time constants for minK38G co-expression averaged 1067 ± 106 ms, versus 1077 ± 129 ms for minK38S (P = ns). Mono-exponential deactivation time constants upon repolarization to –50 mV were also of the same order for minK38G (291 ± 33 ms) and minK38S (246 ± 24 ms, Fig. 1F, n = 20 cells each).

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 A, B: Recordings from transiently transfected CHO cells expressing KvLQT1 and minK38G (A) or minK38S; (B): All data presented in this figure are from 20 CHO cells per group. C: Mean ± S.E.M. IKs–voltage relationships. D: Mean ± S.E.M. normalized tail current/voltage relation fitted by Boltzmann equation (error bars fall within symbols for mean and are therefore not visible). E: Mean ± S.E.M. activation time constants. F: deactivation time constants upon repolarization to –50 mV after a 2-s step to+10 mV. TP=test potential; *P<0.05.

 
Similar results were obtained with COS-7 cells, for which representative recordings are shown in Fig. 2A and B. I_Ks density upon depolarization to +10 mV averaged 44.8 ± 13.7 pA/pF for minK38G vs. 104.2 ± 23.2 pA/pF for minK38S (n = 10, 7 cells, respectively, P<0.05). Activation kinetics at +10 mV were mono-exponential, with time constants averaging 740 ± 114 ms (minK38G) vs. 688 ± 115 ms (minK38S, P = ns), and tail current time constants were also similar: 298 ± 48 (minK38G) vs. 293 ± 39 ms (minK38S, P = ns).

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 A, B: Representative currents recorded from COS-7 cells co-transfected with KvLQT1 and minK38G (A) or minK38S (B). Mean ± S.E.M. IKs-voltage relationships are given in C. TP=test potential; *P<0.05.

 
3.2. Electrophysiological studies–HERG co-expression
Representative currents in COS-7 cells co-transfected with HERG+minK38G or minK38S are shown in Fig. 3A and B. MinK38G co-expression did not obviously alter I_HERG compared to minK38S. Overall mean tail-current densities were similar for the 2 groups (Fig. 3C); e.g. repolarization from a TP of +10 mV evoked I_HERG of 23.1 ± 4.0 pA/pF following co-expression with minK38G, vs. 22.0 ± 5.3 pA/pF upon co-expression with minK38S (n = 12, 15 cells, respectively). Activation V₅₀ was also similar (–9.8 ± 2.7 mV, minK38G vs. –10.1 ± 1.9 mV, minK38S, P = ns). Deactivation kinetics were biexponential and time constants were similar between groups, e.g. repolarizing from +10 mV: _slow averaged 1596 ± 222 ms for minK38G vs. 1345 ± 101 ms for minK38S; _fast averaged 260 ± 37 ms for minK38G vs. 278 ± 35 ms for minK38S (Fig. 3D). There was no difference in the relative contribution of the fast component of deactivation: 38 ± 3% of deactivation occurred with a fast time constant for minK38G vs. 39 ± 3% for minK38S (P = ns).

View larger version (25K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Recordings of IHERG upon co-transfection with minK38G (A) or minK38S (B) in COS-7 cells. C: Mean ± S.E.M. IHERG tail-currents. D: IHERG inactivation kinetics at –50 mV after an activating pulse to+10 mV. (n = 12, 15 cells for HERG+minK38G, HERG+minK38S).

 
3.3. Immunofluorescent studies of KvLQT1–minK interaction
We then evaluated the effect of minK SNP co-expression on KvLQT1 membrane localization. Fig. 4 shows confocal microscope images of double-labelled CHO cells co-transfected with either minK38G (panel A) or minK38S (panel B) and KvLQT1 (both panels). Each panel shows one cell stained for KvLQT1 (upper left), minK (lower left) and the superimposed image (lower right). A dark-field image of the cell is shown at the upper right. Similar results were obtained in seven experiments with each minK variant. Cells co-transfected with minK38G showed less intense 566-nm red-fluorescence (corresponding to TRITC-labelled KvLQT1) at the cell membrane. The 525-nm green fluorescence signal (FITC-labelled minK) was similar between groups. Upon laser line-scanning quantification with identical gain settings, KvLQT1 membrane fluorescence was significantly greater in the presence of minK38S compared with minK38G, whereas minK fluorescence intensity did not differ (Fig. 4C). The membrane KvLQT1/minK fluorescence ratio was approximately twice as great for minK38S compared to minK38G (Fig. 4D), in rough agreement with relative I_Ks densities.

View larger version (42K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Immunofluorescent images of transiently transfected CHO cells double labelled with anti-minK and anti-KvLQT1 antibodies. A, B: Composite images of CHO cells with identical imaging settings. Top left: fluorescence at 566 nm (KvLQT1 secondary label); top right: phase contrast microscopic image; bottom left: fluorescence at 525 nm (minK secondary label); bottom right: superimposed images. C: Mean ± S.E.M. membrane-fluorescence at 566 nm (KvLQT1) and 525 nm (minK). D: KvLQT1/minK intensity ratio. n = 7 per group; *P<0.05; **P<0.005 vs. minK38S.

 
3.4. Biotin–streptavidin assay of KvLQT1–minK interaction
Having observed increased KvLQT1 membrane-localized immunofluorescence with the minK38S isoform, we sought independent confirmation with a different technique. Western blots were performed after surface biotinylation of KvLQT1 and subsequent pulldown with streptavidin-coated beads to isolate membrane proteins. The results were in agreement with the electrophysiological and immunofluorescent studies: co-expression with minK38S increased membrane expression of KvLQT1. Fig. 5 shows a representative blot from transiently transfected CHO cells labelled and extracted with the biotin–streptavidin method. Blotting with anti-KvLQT1 demonstrated a 2.4-fold increase in membrane-associated KvLQT1 protein following co-expression with minK38S (6.8 ± 1.3 arbitrary optical density units, ODU) compared to results following co-transfection with minK38G (2.8 ± 0.5 ODU, n = 5/group, P<0.05).

View larger version (75K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 A representative Western blot obtained after surface biotinylation of KvLQT and pulldown with streptavidin-coated beads. A single specific band was identified at 75 kDa in all samples. The first two lanes show protein isolated from CHO cells co-transfected with KvLQT1 and minK38G, and the last two lanes for cells transfected with KvLQT and minK38S. Similar results were seen in 5 separate experiments with each minK isoform. Molecular weight markers are shown at the left.

 
3.5. Mathematical modeling
To evaluate the potential functional consequences of I_Ks channel density variations of the type seen with the minK SNPs we studied, we used a previously established mathematical model of the human atrial action potential [18] (Fig. 6). To mimic the changes observed in KvLQT1/minK current measurements with the minK38S and 38G isoforms, we set the I_Ks maximal conductance (g_Ks) to 100% and to 50% of its original value, respectively. Panels A and B depict the effect of minK SNPs on the action potential when simulated at 2 Hz with the original model (normal myocyte; panel A) and with I_Kr maximal conductance (g_Kr) set to 0 (to simulate reduced repolarisation reserve; panel B). Under normal conditions, the model for isoform 38G prolonged action potential duration to 90% repolarization (APD₉₀) by 6.2% (Fig. 6A). When the repolarization reserve was reduced (g_Kr=0), APD₉₀ was clearly prolonged to a greater extent (Fig. 6B), by 15.6%. A summary of changes in APD₉₀ due to minK polymorphism under different frequency and repolarization reserve conditions (g_Kr=100%, 50%, or 0%) is presented in panel C. In order to evaluate the potential susceptibility to arrhythmias of a 50% reduction in I_Ks, we sought to determine the minimal pacing frequencies that would create alternans behaviour (at least 1% beat-to-beat variation in APD₉₀). With the basic model, the critical pacing frequencies at which alternans occurred were 3.76 Hz and 3.39 Hz for isoform 38S (g_Ks=100%) and 38G (g_Ks=50%), respectively. When repolarization reserve was reduced by decreasing I_Kr by 50%, the critical frequencies for alternans were reduced to 3.08 Hz for minK38S and 2.65Hz for minK38G. Finally, we evaluated cellular behaviour under specific conditions (g_K1=52.2% control, g_CaL=200% control, g_Kr=0% control, pacing 0.2 Hz) designed to optimize the occurrence of EADs. Under such conditions, EADs were seen for minK38G but not for minK38S (Fig. 6D). Thus, in certain contexts a reduction of I_Ks by 50% could transform a stable response to one in which EADs are generated.

View larger version (26K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Mathematical modeling of the effect of IKs on the human atrial action potential. (A–B) Effect of IKs modulation by the two minK SNPs on the action potential under normal conditions (A) and under reduced repolarization reserve conditions as simulated by the elimination of IKr (B), when activated at 2 Hz. (C) Summary of the relative ADP90 changes with minK38G (as compared with minK38S) at various pacing frequencies for different repolarization reserve conditions. Results are shown up to the highest frequency not associated with alternans. (D) Simulated action potential with a parameter set (gK1=52.2%, gCaL=200%, gKr=0%, and pacing 0.2 Hz) that illustrates the potential of the minK isoforms to determine whether stable or unstable (EAD) action potentials are seen. All results were obtained from the 100th action potential when stimulated from rest at the specified frequency with the Courtemanche–Ramirez–Nattel model. Action potentials plotted with black lines are in the presence of minK38S and dashed lines represent minK38G action potentials.

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
The minK38G/S polymorphism constitutes a common amino acid variation in minK [15]. There is evidence from one relatively small population study that the presence of the minK38G allele is associated with an increased prevalence of AF [16], although this result remains to be confirmed. MinK protein is an accessory subunit crucial for the formation of a normal phenotypic I_Ks in association with the pore-forming -subunit KvLQT1 [19,20], and may also interact with HERG in the formation of I_Kr [21,22]. In the present study, we examined the effects of the minK38G/S polymorphism on currents resulting from KvLQT1 and HERG co-expression, finding significant effects on I_Ks density and KvLQT1 membrane localization/expression but no effects on the properties of I_HERG.

4.1. Potential significance in the context of the role of genetic factors in the pathophysiology of AF
Parental AF increases the risk of the arrhythmia in their offspring [3]. A variety of genetic abnormalities have been reported to predispose to AF, either by altering atrial electrophysiological properties or by the formation of a favourable structural background for the arrhythmia. Potential factors in the pathophysiology of AF include reentry, favoured by abbreviated refractoriness or abnormalities in atrial impulse propagation, or various forms of ectopic impulse formation (in particular early and delayed afterdepolarizations) [23]. Lamin A/C mutations are associated with familial AF, with AF occurring in a context of conduction abnormalities and cardiomyopathy [5,6]. Similarly, Brugada syndrome, which is often caused by loss-of-function mutations in the gene (SCN5A) encoding the cardiac Na⁺ channel and is associated with evidence of conduction abnormalities, may result in AF [8]. Factors that abbreviate atrial repolarization are recognized to promote AF by shortening the wavelength for reentry and favouring the occurrence of multiple wavelet reentry [23]. The short QT syndrome, which can be caused by gain-of-function mutations in HERG [24] or KvLQT1 [25], has been implicated in the causation of AF [9]. In addition, mutations in KvLQT1 [13] and MiRP1 [14] that cause a gain of function in I_Ks are believed to be causal in familial AF, presumably by abbreviating APD and promoting atrial reentry.

There is also evidence for forms of AF that are promoted by afterdepolarization-mediated mechanisms. The ankyrin B mutation implicated in long QT syndrome type 4 causes abnormalities in Ca²⁺ handling and ankyrin B deficiency has been shown to induce both early and delayed afterdepolarizations in mouse cardiomyocytes [11].

Lai and colleagues demonstrated an association between increased AF incidence and a haplotype located on chromosome 21q22 and suggested the KCNE1 gene as a possible candidate [16]. Other genes in the same region were not excluded in this study. However, as I_Ks modulation by a KCNQ1 missense mutation was previously associated with familial AF, the conclusion that alterations in minK might similarly contribute to AF propensity appeared realistic. No functional characterization of this SNP was carried out by Lai and colleagues. As indicated above, the first gene abnormality identified to cause familial AF in the absence of other arrhythmic or structural abnormalities was a KCNQ1 mutation leading to I_Ks gain-of-function [13]. We found the putative AF-associated minK38G isoform to have an opposite effect on I_Ks i.e. reducing current density. Although this seems counterintuitive, there is evidence that APD prolongation can promote atrial tachyarrhythmias in both experimental [26,27] and clinical [12] models. In line with these previous studies, we demonstrate in silico occurrence of EAD with the minK38G SNP in the presence of reduced repolarization reserve, as well as the induction of alternans behaviour at lower frequencies. AF is known to be associated with both long QT [10,11] and short QT [9] syndromes, pointing to potential variability in underlying mechanisms and the possibility that destabilization of repolarization in either direction may be arrhythmogenic.

Our AP modelling studies suggested modest effects on human atrial action potential duration resulting from I_Ks alterations of the order produced in vitro by the SNPs we studied. These effects were enhanced by the reduction of repolarization reserve. The repolarization abnormalities associated with the minK38G polymorphism could produce atrial arrhythmias in 2 ways. The induction of EADs could certainly contribute, although our simulations suggest that quite specific conditions are needed. Another contributor could be the induction of electrical alternans, which is an important pathway to arrhythmia [28] and which occurred at lower frequencies in the presence of minK38G than 38S. It is possible that other abnormalities that impinge on repolarization reserve may unmask the I_Ks discrepancies associated with the 38S/G minK polymorphism and promote AF occurrence. Most patients with long QT syndrome are not diagnosed with AF upon presentation with LQTS and there are no data regarding the potential long-term influence of prolonged atrial action potential duration on the incidence of AF in elderly LQTS patients. It is conceivable that delays in atrial action potential repolarization can lead to slowly developing changes in atrial structure (for example, by increasing atrial Ca²⁺ load) that promote AF indirectly by producing a favourable substrate with aging.

4.2. Potential limitations
The mechanisms of minK–KvLQT1 protein interaction are not completely elucidated. We did not find differences in minK expression at the membrane level with the two isoforms we studied. The increased I_Ks density that we observed upon co-expression with the minK38S isoform might have resulted from more efficient association between KvLQT1 and minK, which facilitated intracellular trafficking of KvLQT1. While changes in I_Ks gating are clearly of great importance in the enhancement of I_Ks current when KvLQT1 is co-expressed with minK [29,30], there is also evidence that alterations in membrane trafficking may occur [31]. The differences in KvLQT1 membrane localization associated with co-expression of the minK38S/G isoforms that we studied points to such a mechanism, which warrants further investigation in future work.

The SNP that we studied is a relatively common one [32], but has not to our knowledge emerged as a risk factor for Torsades de Pointes (TdP) arrhythmias associated with long QT syndromes resulting from ventricular repolarization impairment. The incidence of prolonged QT intervals in minK38G carriers has not been reported [16]. Given the significant differences in I_Ks density between the isoforms that we studied, the lack of an association between minK38G and QT prolongation or TdP risk would be surprising. It is possible that the role of minK in KvLQT1 trafficking in native cells is different from that in the two cell lines we examined, or that the large differences that we observed require overexpression of both minK and KvLQT1, as occurs in heterologous expression systems. Another possibility is that the minK38G/S polymorphism is inherited as part of a haplotype, a common means of transmission in which multiple SNPs occur over a finite chromosomal region and are inherited in a block. Associated, co-inherited SNPs in the minK38G block could conceivably contribute importantly to the electrophysiological consequences of the haplotype, a possibility that we hope will be examined further in the light of our findings.

	5. Conclusions

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 
We have performed the first detailed characterization of the effects of the minK38G/S polymorphism on currents resulting from co-expression with delayed-rectifier -subunits. Our results suggest that minK38S co-expression results in larger I_Ks currents and greater KvLQT1 membrane expression than minK38G. Action potential effects predicted by a computer model of the human atrial action potential were subtle, but were enhanced in the presence of destabilized repolarization due to impaired repolarization reserve and provide potential insights into physiological bases for the minK38G-AF association. Further genetic studies of the relationship between the minK38S/G polymorphism and arrhythmia susceptibility, as well as of any associated polymorphisms, would seem warranted.

	Acknowledgements

 
We thank Louis Villeneuve for excellent technical assistance and France Thériault for secretarial help. The study was supported by the Canadian Institutes of Health Research, the Quebec Heart and Stroke Foundation and the Mathematics of Information Technology and Complex Systems (MITACS) Network of Centers of Excellence. Joachim R. Ehrlich was a Heart and Stroke Foundation of Canada (HSFC) Fellow. Stephen Zicha was supported by a "Fonds de la recherche en santé du Québec" studentship. Terence E. Hébert is a McDonald Scholar of the Heart and Stroke Foundation of Canada.

	Notes

 
¹ Both authors contributed equally to this work.

Time for primary review 38 days

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion 5. Conclusions References

 

1. Darbar D., Herron K.J., Ballew J.D., Jahangir A., Gersh B.J., Shen W.K., et al. Familial atrial fibrillation is a genetically heterogeneous disorder. J Am Coll Cardiol (2003) 41:2185–2192.[Abstract/Free Full Text]
2. Ellinor P.T., MacRae C.A. The genetics of atrial fibrillation. J Cardiovasc Electrophysiol (2003) 14:1007–1009.[CrossRef][Medline]
3. Fox C.S., Parise H., D'Agostino R.B. Sr., Lloyd-Jones D.M., Vasan R.S., Wang T.J., et al. Parental atrial fibrillation as a risk factor for atrial fibrillation in offspring. JAMA (2004) 291:2851–2855.[Abstract/Free Full Text]
4. Brugada R., Tapscott T., Czernuszewicz G.Z., Marian A.J., Iglesias A., Mont L., et al. Identification of a genetic locus for familial atrial fibrillation. N Engl J Med (1997) 336:905–911.[Abstract/Free Full Text]
5. Garg A., Speckman R.A., Bowcock A.M. Multisystem dystrophy syndrome due to novel missense mutations in the amino-terminal head and alpha-helical rod domains of the lamin A/C gene. Am J Med (2002) 112:549–555.[CrossRef][Web of Science][Medline]
6. Sebillon P., Bouchier C., Bidot L.D., Bonne G., Ahamed K., Charron P., et al. Expanding the phenotype of LMNA mutations in dilated cardiomyopathy and functional consequences of these mutations. J Med Genet (2003) 40:560–567.[Abstract/Free Full Text]
7. Tsai C.T., Lai L.P., Lin J.L., Chiang F.T., Hwang J.J., Ritchie M.D., et al. Renin–angiotensin system gene polymorphisms and atrial fibrillation. Circulation (2004) 109:1640–1646.[Abstract/Free Full Text]
8. Morita H., Kusano-Fukushima K., Nagase S., Fujimoto Y., Hisamatsu K., Fujio H., et al. Atrial fibrillation and atrial vulnerability in patients with Brugada syndrome. J Am Coll Cardiol (2002) 40:1437–1444.[Abstract/Free Full Text]
9. Gaita F., Giustetto C., Bianchi F., Wolpert C., Schimpf R., Riccardi R., et al. Short QT Syndrome: a familial cause of sudden death. Circulation (2003) 108:965–970.[Abstract/Free Full Text]
10. Schott J.J., Charpentier F., Peltier S., Foley P., Drouin E., Bouhour J.B., et al. Mapping of a gene for long QT syndrome to chromosome 4q25–27. Am J Hum Genet (1995) 57:1114–1122.[Web of Science][Medline]
11. Mohler P.J., Schott J.J., Gramolini A.O., Dilly K.W., Guatimosim S., duBell W.H., et al. Ankyrin-B mutation causes type 4 long-QT cardiac arrhythmia and sudden cardiac death. Nature (2003) 421:634–639.[CrossRef][Medline]
12. Kirchhof P., Eckardt L., Franz M.R., Monnig G., Loh P., Wedekind H., et al. Prolonged atrial action potential durations and polymorphic atrial tachyarrhythmias in patients with long QT syndrome. J Cardiovasc Electrophysiol (2003) 14:1027–1033.[CrossRef][Web of Science][Medline]
13. Chen Y.H., Xu S.J., Bendahhou S., Wang X.L., Wang Y., Xu W.Y., et al. KCNQ1 gain-of-function mutation in familial atrial fibrillation. Science (2003) 299:251–254.[Abstract/Free Full Text]
14. Yang Y., Xia M., Jin Q., Bendahhou S., Shi J., Chen Y., et al. Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation. Am J Hum Genet (2004) 75:899–905.[CrossRef][Web of Science][Medline]
15. Lai L.P., Deng C.L., Moss A.J., Kass R.S., Liang C.S. Polymorphism of the gene encoding a human minimal potassium ion channel (minK). Gene (1994) 151:339–340.[CrossRef][Web of Science][Medline]
16. Lai L.P., Su M.J., Yeh H.M., Lin J.L., Chiang F.T., Hwang J.J., et al. Association of the human minK gene 38G allele with atrial fibrillation: evidence of possible genetic control on the pathogenesis of atrial fibrillation. Am Heart J (2002) 144:485–490.[CrossRef][Web of Science][Medline]
17. Gerald C.F., Wheatley P.O. Applied numerical analysis. (1984) Reading, Mass.: Addison-Wesley Pub.
18. Courtemanche M., Ramirez R.J., Nattel S. Ionic mechanisms underlying human atrial action potential properties: insights from a mathematical model. Am J Physiol (1998) 275:H301–H321.[Web of Science][Medline]
19. Sanguinetti M.C., Curran M.E., Zou A., Shen J., Spector P.S., Atkinson D.L., et al. Coassembly of K(V)LQT1 and minK (IsK) proteins to form cardiac I(Ks) potassium channel. Nature (1996) 384:80–83.[CrossRef][Medline]
20. Barhanin J., Lesage F., Guillemare E., Fink M., Lazdunski M., Romey G. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature (1996) 384:78–80.[CrossRef][Web of Science][Medline]
21. Yang T., Kupershmidt S., Roden D.M. Anti-minK antisense decreases the amplitude of the rapidly activating cardiac delayed rectifier K+°current. Circ Res (1995) 77:1246–1253.[Abstract/Free Full Text]
22. Ohyama H., Kajita H., Omori K., Takumi T., Hiramoto N., Iwasaka T., et al. Inhibition of cardiac delayed rectifier K+ currents by an antisense oligodeoxynucleotide against IsK (minK) and over-expression of IsK mutant D77N in neonatal mouse hearts. Pflugers Arch (2001) 442:329–335.[CrossRef][Web of Science][Medline]
23. Nattel S. New ideas about atrial fibrillation 50 years on. Nature (2002) 415:219–226.[CrossRef][Medline]
24. Brugada R., Hong K., Dumaine R., Cordeiro J., Gaita F., Borggrefe M., et al. Sudden death associated with short-QT syndrome linked to mutations in HERG. Circulation (2004) 109:30–35.[Abstract/Free Full Text]
25. Bellocq C., van Ginneken A.C., Bezzina C.R., Alders M., Escande D., Mannens M.M., et al. Mutation in the KCNQ1 gene leading to the short QT-interval syndrome. Circulation (2004) 109:2394–2397.[Abstract/Free Full Text]
26. Satoh T., Zipes D.P. Cesium-induced atrial tachycardia degenerating into atrial fibrillation in dogs: atrial torsades de pointes? J Cardiovasc Electrophysiol (1998) 9:970–975.[Web of Science][Medline]
27. Burashnikov A., Antzelevitch C. Reinduction of atrial fibrillation immediately after termination of the arrhythmia is mediated by late phase 3 early afterdepolarization-induced triggered activity. Circulation (2003) 107:2355–2360.[Abstract/Free Full Text]
28. Li M., Otani N.F. Controlling alternans in cardiac cells. Ann Biomed Eng (2004) 32:784–792.[CrossRef][Web of Science][Medline]
29. Tapper A.R., George A.L. Jr. MinK subdomains that mediate modulation of and association with KvLQT1. J Gen Physiol (2000) 116:379–390.[Abstract/Free Full Text]
30. Tapper A.R., George A.L. Jr. Location and orientation of minK within the I(Ks) potassium channel complex. J Biol Chem (2001) 276:38249–38254.[Abstract/Free Full Text]
31. Krumerman A., Gao X., Bian J.S., Melman Y.F., Kagan A., McDonald T.V. An LQT mutant minK alters KvLQT1 trafficking. Am J Physiol (Cell Physiol) (2004) 286:C1453–C1463.[Abstract/Free Full Text]
32. Ackerman M.J., Tester D.J., Jones G.S., Will M.L., Burrow C.R., Curran M.E. Ethnic differences in cardiac potassium channel variants: implications for genetic susceptibility to sudden cardiac death and genetic testing for congenital long QT syndrome. Mayo Clin Proc (2003) 78:1479–1487.[Abstract/Free Full Text]

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