Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone...

557Biomarkers Med. (2014) 8(4), 557–570 ISSN 1752-0363

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Aims: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Disturbances in cardiac potassium conductance are considered as one of the disease mechanisms in AF. We aimed to investigate if mutations in potassium-channel β-subunits KCNE2 and KCNE3 are associated with early-onset lone AF. Methods & results: The coding regions of KCNE2 and KCNE3 were bidirectionally sequenced in 192 unrelated patients diagnosed with early-onset lone AF (<40 years). Two nonsynonymous missense mutations were identified in KCNE2 (M23L and I57T). Both mutations were absent in a healthy control group (n = 1500 alleles). Electrophysiological investigations were performed for both mutations in combination with candidate pore-forming α-subunits KV7.1, KV11.1, KV4.3 and KV1.5. A significant gain-of-function effect was observed upon coexpression with KV7.1 and KV7.1 + KCNE1. Confocal imaging found no differences in subcellular localization. No disease-suspected mutations were identified in KCNE3. Conclusion: We identified two KCNE2 gain-of-function missense mutations that seem to increase the susceptibility of early-onset lone AF. These results confirm previous findings indicating that gain-of-function in the slow delayed rectifier potassium current might be involved in the pathogenesis of AF.

Keywords: atrial fibrillation • genetics • I57T • KCNE2 • KCNE3 • KV7.1 • M23L • R83H

Cardiac voltage-gated potassium channels are dynamic macromolecular structures consist-ing of pore-forming α-subunits assembled with a variety of accessory β-subunits, the cytoskeleton and various signaling complexes. These potassium channel complexes are essential for cardiac action potential repolar-ization and functional changes in these struc-tures might lead to impaired electrical and contractile properties of the heart. Genetic mutations in cardiac voltage-gated potassium channels and accessory proteins have been shown to underlie various cardiac arrhythmias including atrial fibrillation (AF) [1,2].

AF is characterized by rapid and irregular electrical activation of the atrium and is the most frequent sustained cardiac arrhythmia, occurring in approximately 1% of the general population. The arrhythmia is responsible for significant morbidity and mortality, pri-marily because of an increased risk of stroke

and heart failure [3]. Increasing evidence supports that AF has a considerable familial component [4], and this heredity seems to be most significant in patients with onset of the disease at a young age and in the absence of predisposing diseases such as hypertension, ischemic and/or valvular heart diseases [5]. Such individuals with onset of AF at a young age and without concomitant disease are designated as having ‘lone’ AF.

A prevailing conceptual model for AF describes reduced electrical refractoriness as a substrate for re-entrant arrhythmia [6]. In support of this paradigm, several gain-of-function mutations in genes related to potas-sium conductance and involved in cardiac repolarization have been associated with AF (KCNQ1, KCNH2, KCNE1,2,3,5, KCNA5 and KCND3) [7–14]. Most convincingly, this has been shown for the gene KCNQ1 encod-ing the pore-forming α-subunit of the slow

Jonas Bille Nielsen‡,1,2, Bo Hjorth Bentzen‡,1,3, Morten Salling Olesen1,2, Jens-Peter David1,3, Søren-Peter Olesen1,3, Stig Haunsø1,2,4, Jesper Hastrup Svendsen1,2,4 & Nicole Schmitt*,1,3

1Danish National Research Foundation

Centre for Cardiac Arrhythmia (DARC),

Copenhagen, Denmark 2Laboratory for Molecular Cardiology,

The Heart Centre, Rigshospitalet,

University of Copenhagen, Denmark 3Department of Biomedical Sciences,

Faculty of Health & Medical Sciences,

University of Copenhagen, Denmark 4Department of Medicine & Surgery,

Faculty of Health Sciences, University of

Copenhagen, Denmark

*Author for correspondence:

Tel.: +45 35327448

[email protected]

Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation

Research Article

10.2217/BMM.13.137 © 2014 Future Medicine Ltd

Biomarkers Med.

10.2217/BMM.13.137

Research Article

NielseN, BeNtzeN, OleseN et al.

Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation

8

4

2014

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558 Biomarkers Med. (2014) 8(4)

delayed rectifier (IKs

) current [7]. However, the major-ity of these potassium-channel related genes have only sporadically been linked to AF [15].

Both KCNE2, encoding the KCNE2 (MiRP1) protein, and KCNE3, encoding KCNE3 (MiRP2), are expressed in human atrial tissue and act as regu-latory β-subunits of different cardiac potassium cur-rents, including rapid delayed rectifier current (I

Kr) and

IKs

[16–18]. Despite the fact that KCNE2, and to some extent KCNE3, are considered susceptibility genes in AF, only one family-specific mutation in each of these two genes has, to the best of our knowledge, so far been associated with AF [10,11]. We analyzed a highly selected cohort of patients with onset of lone AF at a very early age, as such individuals are believed to be more likely to have a primary genetic defect as a substrate for AF. These individuals were investigated for genetic variants in KCNE2 and KCNE3 as possible substrates for their early-onset AF.

MethodsStudy subjectsPatients with onset of lone AF (i.e., absence of clinical or echocardiographic findings of other cardiovascular diseases, hypertension, metabolic, or pulmonary dis-eases) who were <40 years of age were consecutively included from eight hospitals in the Copenhagen region of Denmark [19]. Additionally, we included two control populations consisting of ethnically matched individuals free of heart disease as described previously [13,20]. The study was approved by the Scientific Ethics Committee of Copenhagen and Frederiksberg (Proto-col reference number: KF 01313322), and conforms to the principles outlined in the Declaration of Helsinki. All included subjects gave written informed consent.

Mutation screeningGenomic DNA was extracted from blood samples using the QIAamp DNA Blood Mini Kit (QIAGEN, Hilden, Germany). Oligonucleotide primers for exons and splice junctions were designed using the known sequence of human KCNE2 (GenBank Acc. No. NM_172201.1) or KCNE3 (NM_005472.4), respec-tively. Primers were designed with M13 tail sequences. DNA fragments were amplified by touchdown PCR and directly sequenced using M13 primers and Big Dye chemistry (DNA analyzer 3730; Applied Biosys-tems, CA, USA). The identified variants were validated by resequencing of a second PCR product.

To rule out contribution to AF from genes previ-ously associated with AF, bidirectional sequencing of the coding regions of KCNQ1 (NM_000218.2), KCNH2 (NM_000238.2), SCN5A (NM_198056.2), KCNA5 (NM_002234.2), KCNE1 (NM_000219.3),

KCNE5 (NM_012282.2), KCNJ2 (NM_000891.2), KCNJ5 (NM_000890.3), SCN1–3B (NM_199037.4, NM_004588.4, NM_001040151.1), KCNN3 (NM_002249.4), LMNA (NM_005572) and NPPA (NM_006172.3) was performed.

BioinformaticsdbSNP [21] and NHLBI Exome Sequencing Project (ESP) Exome Variant Server [22] was accessed for evalu-ation of the prevalence of the identified mutations in the general population. PolyPhen-2 [23] was used to pre-dict the possible impact of an amino acid substitution on the structure and function of the protein [24].

Molecular biologyThe point mutations M23L (c.67A>T) and I57T (c.170T>C) in KCNE2 were introduced using mutated oligonucleotide extension (PfuTurbo Polymerase; Stratagene, CA, USA) from the plasmid template harboring the coding sequence of hKCNE2, digested with DpnI (Fermentas, St. Leon-Roth, Germany) and transformed into Escherichia coli XL1 Blue cells. All plasmids were verified by complete DNA sequencing of the cDNA insert (Macrogen Inc., Seoul, Republic of Korea). cRNA was prepared from linearized plas-mids using the mMESSAGEmMACHINE T7 kit (Ambion, TX, USA) according to the manufacturer’s instructions and stored at -80°C until injection. RNA concentrations were quantified by UV spectroscopy and quality was checked by gel electrophoresis.

Confocal microscopy & imagingTransiently transfected CHO-K1 cells grown on glass coverslips were fixed in 4% paraformaldehyde in phos-phate buffered saline (PBS) for 15 min at room tem-perature. Blocking and permeabilization was performed by a 30-min incubation with 0.2% fish skin gelatin in PBS supplemented with 0.1% Triton X-100 (PBST). The cells were incubated for 1 h in primary antibodies diluted in PBST, followed by 45-min incubation with secondary antibodies (in PBST). The coverslips were mounted in Prolong Gold (Invitrogen, Glostrup, Den-mark). Phalloidin was used to stain F-actin filaments and therefore used as a plasma membrane marker.

Laser scanning confocal microscopy was performed using the Zeiss LSM 780 confocal system (Martin-sried, Germany) . Images were acquired using a 63× oil immersion objective, NA 1.4 with a pinhole size of 1 AU and a pixel format of 1024 × 1024. Line averaging was used to reduce noise. Sequential scanning was employed to allow the separation of signals from the individual channels. The images were treated using the Zen 2009 software (Zeiss) and Adobe Photoshop CS5 (CA, USA).

The following antibodies were used: Goatpolyclonal

future science group

Research Article Nielsen, Bentzen, Olesen et al.

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anti-KV7.1 (2 μg/ml, C-20; Santa Cruz Biotechnol-

ogy, Heidelberg,Germany), rat monoclonal anti-HA (2 μg/ml, clone 3F10; Roche, IN, USA), Alexa-Fluor 488 donkey anti-goat IgG (10 μg/ml), Alexa-Fluor 488 donkey anti-rat IgG (10 μg/ml), Alexa-Fluor 555 don-key anti-goat IgG (4 μg/ml), Alexa-Fluor 647 phal-loidin (1.5 U/ml), rhodamine-conjugated phalloidin (1.5 U/ml). All Alexa Fluor®-coupled reagents and phalloidin were from Invitrogen (Glostrup, Denmark).

Two-electrode voltage clampXenopus laevis oocytes were purchased from EcoCyte Bioscience (Castrop-Rauxel, Germany). Oocytes were kept at 19°C and currents were measured 3 days after injection of cRNA of the ion channel subunits in 1:1 molar ratios. For K

V7.1 + KCNE1 an additional set of

experiments using a 1:4:4 (KV7.1:KCNE1:KCNE2)

molar ratio was conducted. Recordings from oocytes were performed using a two-electrode voltage-clamp amplifier (Dagan CA-1B; IL, USA).

Borosilicate glass recording electrodes (Module Ohm, Herlev, Denmark) were made using a DMZ-Universal Puller (Zeitz Instruments, Martinsried, Ger-many) and had a resistance of 0.5–1 MΩ when filled with 2 M KCl. Oocytes were superfused with Kulori solution (NaCl 90 mm, KCl 4 mm, MgCl

2 1 mm,

CaCl2 1 mm, 4-(2-hydroxyethyl)-1-piperazineethane-

sulfonic acid 5 mm, pH = 7.4 with NaOH) and experi-ments were performed at room temperature. Data acquisition was performed with the Pulse software (HEKA Elektronik, Lambrecht/Pfalz, Germany). For all recordings, the holding potential was -80 mV.

Data analysisData analysis was performed with Igor Pro (Wave-metrics, OR, USA) and GraphPad Prism (GraphPad Software Inc., CA, USA). A Boltzmann function:

was fitted to activation and inactivation curves to obtain the potential of half-maximal activation/inacti-vation (V

50) and the slope factor (k). Data are repre-

sented as mean ± standard error of the mean, unless otherwise indicated. Two-way analysis of variance sta-tistical analyses followed by a Bonferroni post-test or analysis of variance followed by Tukey’s method of mul-tiple comparisons were used as appropriate to compare the wild-type and mutated KCNE2 channel complex. A two-sided p-value <0.05 was considered statistically significant.

ResultsStudy cohortThe study population consisted of 192 patients with onset of AF ranging from 16 to 39 years, without any concomitant disease. The two control popula-tions comprised 750 individuals free of heart disease. All included individuals were of Danish/Caucasian ethnicity. Clinical data of the study population have been described earlier [19] and are shown in Table 1.

Mutation screening & bioinformaticsDirect DNA sequencing of KCNE2 in 192 index patients of Danish/Caucasian ethnicity revealed two nonsynonymous mutations: c.67A>T, lead-ing to replacement of methionine with leucine at codon 23 (M23L); and c.170T>C, where isoleucine was replaced with threonine at position 57 (I57T; Figure 1). Neither of the mutations was found in the control population (n = 1500 alleles) and has not pre-viously been reported to be associated with AF. M23L was also absent in 13,006 alleles from individuals of mixed ethnicity on the ESP exome server. I57T was present in two out of 8600 alleles from individuals of European–American ethnicity and in three out of 4406 alleles from individuals of African–American ethnicity.

Both genetically affected probands were hetero-zygous carriers. Positions M23 and I57 in KCNE2 are both highly conserved across species suggesting func-tional importance (Figure 1). Both M23L and I57T


Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation Research Article

Table 1. Clinical characteristics of the lone atrial fibrillation population (n = 192).

Parameter Value

Median age of onset; years (IQR) 31.5 (26–36)

Male gender (%) 82

Height (cm) 183 ± 9

Weight (kg) 89 ± 17

BMI (kg/m2) 26.7 ± 4.6

Blood pressure (mmHg)

Systolic 131 ± 13

Diastolic 78 ± 9

AF type (%)

Paroxysmal 55.9

Persistent 35.9

Permanent 8.2

Family history of AF

First-degree relatives with AF (%) 31

All numbers are reported as mean ± standard deviation unless

otherwise noted.

AF: Atrial fibrilliation; IQR: Interquartile range.


were predicted as probably damaging by PolyPhen-2 with the scores 0.962 and 0.970, respectively.

Screening of KCNE3 identified one nonsynonymous variant: c.248G>A, leading to an exchange of argi-nine to histidine at position 83 (R83H). This variant, however, was also found in one healthy individual in the control population and R83H was also reported in 58 out of 8586 (0.7%) alleles from individuals of European–American ethnicity on the ESP exome server.

Clinical dataProband no. 1 carrying the mutation M23L (male, age 37) was diagnosed with paroxysmal AF at the age of 26 years. The frequency of AF was higher during periods of physiological stress. The patient had a sinus rhythm ECG with discrete J-waves in I, II, aVF and V5. There was no sign of hypertrophy or ischemia, and the ECG was otherwise normal (PR 154 ms, QRS 96 ms, QTc 419 ms). An echocardiography was performed without any remarks (left ventricular ejection fraction 65%; left atrium <40 mm).

Proband no. 2 carrying the mutation I57T (male, age 46 years) had experienced episodes of palpita-

tions and chest discomfort since the age of 17 years and he was diagnosed with paroxysmal AF at age of 36 years. The patient had a normal sinus rhythm ECG (PR 145 ms, QRS 90 ms, QTc 389 ms) and also the echocardiography was normal.

Neither of the two patients had ever experienced syncope or near syncope. Both patients had normal blood tests, including thyroid stimulating hormone within the normal range. Neither of the probands had a familial history of AF. They were both free of mutations in genes previously related to AF (see the ‘Methods’ section).

Functional characterization of the KCNE2 mutationsEffect on IKs

KCNE2 has been shown to interact with KV7.1

[25,26]. Hence, we tested the effect of KCNE2 wild-type (WT) or mutants on currents elicited by K

V7.1

(Figure 2) applying a voltage step protocol from -80 mV to +60 mV in 20 mV increments for 2 s. Coex-pression of KCNE2-WT with K

V7.1 in Xenopus lae-

vis oocytes generated currents with decreased current



Figure 1. Clinical characterization and genetic analysis of probands. (A) DNA sequence analysis revealing the two mutations M23L and I57T (see text). (B) KCNE2 species alignment for position M23 and I57, respectively, showing that both mutation sites were very well conserved among species. (C) Position of the mutations indicated in schematic of protein topology. (D) ECG trace from patient no. 1 carrying the mutation M23L showing atrial fibrillation. (E) ECG trace from patient no. 2 (I57), here illustrated by a sinus rhythm QRS complex from lead V1–V3, showing no sign of Brugada syndrome ECG patterns. The QTc interval was 389 ms. Both ECG traces were recorded at a paper speed of 25 mm/s and the voltage was calibrated at 1 mV/mm. For color images please see online www.futuremedicine.com/doi/pdf/10.2217/bmm.13.137 †Identical. ‡Conserved. §Variable.

KCNE2 M23L KCNE2 I57T

KCNE2 M23L I57T

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www.futuremedicine.com 561future science group


Figure 2. Effect of KCNE2 wild-type or KCNE2 mutants on KV7.1 currents. (A) Representative current traces recorded at test potentials from -80 mV to +60 mV from Xenopus laevis oocytes expressing KV7.1 alone (upper left) or when coexpressed with wild-type KCNE2 (upper right), KCNE2-M23L (lower left) or KCNE1-I57T (lower right). (B) To construct current–voltage relationships, currents were elicited using the voltage-clamp protocol. Steady-state currents measured at the end of each step, were normalized to the maximum current measured from X. laevis oocytes expressing KV7.1 alone, and plotted as a function of the test potential. Two-way analysis of variance statistical analyses followed by a Bonferroni post-test were performed to compare the effect of the KCNE2 mutant on the current–voltage relationship: KV7.1+KCNE2 versus KV7.1 (†); KV7.1+KCNE2 versus KV7.1+KCNE2-M23L (‡); KV7.1+KCNE2 versus KV7.1+KCNE2-I57T (§). (C) Voltage dependence of activation was investigated by plotting the normalized peak tail current measured at -30 mV against the preceding step potential. V50 values were calculated from individual Boltzmann fits to the normalized tail current–voltage relationships. One way analysis of variance analysis followed by a Turkey’s multiple comparison test was performed to investigate the effect of KCNE2 mutations on V50 (KV7.1+KCNE2 vs KV7.1 [p > 0.05]; KV7.1+KCNE2 vs KV7.1+KCNE2-M23L [p < 0.01]; KV7.1+KCNE2 vs KV7.1+KCNE2-I57T [p < 0.01]. KV7.1, KV7.1+KCNE2; KV7.1+KCNE2-M23L, and KV7.1+KCNE2-I57T: n = 21, 22, 17, and 20, respectively). Vm: Membrane potential.

Kv7.1

Kv7.1+KCNE2-I57T

Kv7.1+KCNE2

Kv7.1+KCNE2-M23L

2 µA

1 s

Vm (mV)

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ax

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Kv7.1

Kv7.1+KCNE2-M23L

Kv7.1+KCNE2-I57T

‡

†††

§§§‡‡‡

†††

§§§‡‡‡

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density as reported earlier [27]. We have previously shown that effects were comparable using different expression systems [28]. Upon coexpression of the mutant β-subunits, current levels were similar to lev-els when K

V7.1 was expressed alone indicating both

mutations led to a gain-of-function – that is, higher current amplitudes. Representative traces are shown in Figure 2A. The data is summarized in current–volt-age relationships depicted in Figure 2B. Steady-state currents measured at the end of each step were nor-malized to the maximum current measured from X. laevis oocytes expressing K

V7.1 alone, and plotted as

a function of the test potential showing significant gain-of-function for both M23L and I57T. The volt-age dependence of activation was investigated by plot-ting the normalized peak tail current measured at -30 mV against the preceding step potential (Figure 2C). V

50-values calculated from individual Boltzmann fits

to the normalized tail current–voltage relationships demonstrated that the KCNE2 mutations led to a sig-nificant negative shift of V

50 as compared with K

V7.1

coexpressed with KCNE2.

To analyze whether the changes in current observed when coexpressing the mutant β-subunits were caused by altered subcellular localization of the β-subunits, we expressed WT or mutant KCNE2 in CHO-K1 cells and performed confocal imaging (Figure 3). When expressed alone, mutant β-subunits localized to the both Golgi compartment and the plasma membrane similar to WT KCNE2. Surface membrane localiza-tion was visible as overlap with the membrane marker Phalloidin (Figure 3A). Upon coexpression with K

V7.1,

α- and β-subunits colocalized at the cell membrane surface. This colocalization was not affected by the mutations (Figure 3B).

Since KCNE2 has been ascribed a role in the genera-tion of I

Ks [27] that is composed of K

V7.1 and KCNE1

we next investigated the effect of KCNE2 WT or mutants on I

Ks . Figure 4A shows representative current

traces obtained upon a voltage step protocol from -80 mV to +60 mV with 5 s voltage steps. Coexpression of KCNE1 led to a slowing of channel activation and increased current amplitude as reported earlier [29,30]. When K

V7.1/KCNE1/KCNE2 were coexpressed in



Figure 3. Effect of KCNE2 wild-type or KCNE2 mutants on subcellular localization. (A) Mutations M23L and I57T in KCNE2 do not disturb the localization of the subunit in CHO-K1 cells: HA-tagged versions of KCNE2, KCNE2-M23L and KCNE2-I57T were singly expressed in CHO-K1 cells (i, ii & iii, respectively). Each subunit was capable of trafficking to the plasma membrane where they colocalized with Pha, which was used as a membrane marker. White arrows exemplify colocalization in the merged pictures. (B) KCNE2-M23L and KCNE2-I57T colocalize at the plasma membrane with (i) KV7.1: KV7.1 (red) was able to traffic to the plasma membrane when singly expressed in CHO-K1 cells. (ii–iv) When KV7.1 was coexpressed together with each HA-tagged version of KCNE2, KCNE2-M23L and KCNE2-I57T, respectively, colocalization was observed at the plasma membrane (Pha) using confocal microscopy. Sequential scanning was used to reduce crosstalk. Merged images are shown in the right column and white arrows indicate colocalization at the plasma membrane. For color images please see online www.futuremedicine.com/doi/pdf/10.2217/bmm.13.137 Pha: Phalloidin.

Bi

ii

iii

iv

HA-E2 Pha Merge

HA-E2-M23L Pha Merge

HA-E2-I57T Pha Merge

HA-E2

HA-E2-M23L

HA-E2-I57T

Kv7.1

Kv7.1

Kv7.1

Kv7.1

Pha

Pha

Pha

Pha

Merge

Merge

Merge

Merge

20 µm

20 µm

i

iii

iv

ii

i

iii

ii



Figure 4. Effect of KCNE2 wild-type or KCNE2 mutants on the slow delayed rectifier current. (A) Representative current traces recorded as in Figure 2: KV7.1+KCNE1 alone (left) or when coexpressed with wild-type KCNE2 (right), the voltage-clamp protocol is shown in the inset in (B). (B & C) Steady-state currents measured at the end of each step were normalized to the maximum current measured from Xenopus laevis oocytes expressing KV7.1+KCNE1 alone, and plotted as a function of the test voltage (left). Voltage dependence of activation was investigated by plotting the normalized peak tail current measured at -30 mV against the preceding step potential. The normalized data were fitted to a two-state Boltzmann distribution (right). In (B) oocytes were injected with KV7.1, KCNE1 and KCNE2 in a 1:1:1 molar ratio, while in (C) oocytes were injected in a 1:4:4 molar ratio. Two-way analysis of variance statistical analyses followed by a Bonferroni post-test were performed to compare the effect of the KCNE2 mutant on the current–voltage relationship: KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1 (†); KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1+KCNE2-M23L (‡); KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1+KCNE2-I57T (§). A one-way analysis of variance analysis followed by a Turkey’s multiple comparison test was performed to investigate the effect of KCNE2 mutations on V50 (KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1 [p > 0.05]; KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1+KCNE2-M23L [p < 0.01]; KV7.1+KCNE1+KCNE2 versus KV7.1+KCNE1+KCNE2-I57T [p < 0.05]. KV7.1+KCNE1, KV7.1+KCNE1+KCNE2, KV7.1+KCNE1+KCNE2-M23L and KV7.1+KCNE1+KCNE2-I57T: (B) n = 20, 17, 18 and 14, respectively; (C) n = 12, 12 and 12, respectively). Vm: Membrane potential.

1 s

5 µA

Kv7.1+KCNE1 Kv7.1+KCNE1+KCNE2

No

rmal

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cu

rren

tN

orm

aliz

ed c

urr

ent

Vm (mV)

Vm (mV) Vm (mV)

Vm (mV)

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ail m

axI T

ail/I

Tai

l max

60 mV

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-30 mV

5 s

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Kv7.1+KCNE1 Kv7.1+KCNE1+KCNE2 Kv7.1+KCNE1+KCNE2-I57LKv7.1+KCNE1+KCNE2-M23L

‡‡‡§

†††‡‡‡§§


a 1:1:1 molar ratio, the channel complex resulted in slightly smaller currents with similar activation prop-erties as the K

V7.1/KCNE1 in line with reports by Wu

et al. [27]. Coexpression and analysis of the mutants’ normalized current–voltage relationship (Figure 4B) or voltage dependence of activation (Figure 4B) revealed that they did not exert an effect on I

Ks. However, when

the amount of KCNE2 cRNA coexpressed with KV7.1

and KCNE1 was increased, the mutant KCNE2 chan-nel complexes displayed a gain-of-function as compared with the WT KCNE2 channel complex, demonstrated by a significant increase in steady-state current ampli-tude and a significant hyperpolarizing shift in the volt-age dependence of channel activation (V

50: KCNE2-

WT = 23 ± 3 mV; KCNE2-M23L = 12.4 ± 2 mV; KCNE2-I57T = 14 ± 2 mV; Figure 4C).

Effect on IKr

KV11.1 is the pore-forming subunit of the I

Kr cur-

rent, which is crucial for termination of the cardiac action potential [31]. KCNE2 has also been reported to interact with K

V11.1 affecting both peak current

densities and biophysical parameters [32]. We coex-pressed KCNE2 and its mutants with K

V11.1 in X.

laevis oocytes and elicited current by applying a volt-age step protocol stepping from a holding potential of -80 mV to -60 mV to +40 mV for 2 s; tail cur-rents were recorded at -60 mV (Figure 5). As expected, K

V11.1 alone passed little outward current upon depo-

larization, but conducted large currents upon repolar-ization back to negative potentials. In contrast with previous results we found KCNE2 to increase current densities, albeit changes were not significant [32]. Rep-resentative traces are shown in Figure 5A, and the cor-responding typical bell-shaped current–voltage rela-tionships in Figure 5B. The KCNE2 mutants M23L and I57T also increased K

V11.1 current amplitudes

similar to WT, with a trend of M23L towards more current compared with KCNE2-WT, yet not reach-ing significance. Voltage dependence of activation was investigated by plotting the normalized peak tail cur-rent measured at -60 mV against the preceding step potential (Figure 5C), no difference between WT and mutants was observed. We also investigated the volt-age-dependent release from inactivation (Figure 5C, lower panel), yet we did not observe any difference between WT and mutants.

Effect on the transient outward currentIn another attempt to reveal the impact of these novel KCNE2 mutations, we analyzed the effect of KCNE2 on transient outward current (I

to) that is present in both

atria and ventricles. Native Ito is considered to be com-

posed of KV4.3, KChIP2 and possibly other subunits

[33]. Also KCNE2 has been reported to interact with K

V4.3 [34,35]. Using heterologous expression in mam-

malian HEK293 cells, Wu and colleagues have shown that KCNE2 increased amplitudes of K

V4.3+KChIP2

currents [35]. We expressed Ito in the absence and

presence of KCNE2 in X. laevis oocytes (Figure 6). Representative current traces recorded at test poten-tials from -60 mV to +40 mV from oocytes express-ing K

V4.3+KChIP2 alone (left) or when coexpressed

with KCNE2-WT (right) are shown in Figure 6A. In line with published data, we observed an increase in current amplitudes summarized in the IV curves depicted in Figure 6B [35]. Steady-state currents at the end of the step pulse were normalized to the maximum current measured from X. laevis oocytes expressing K

V4.3+KChIP2 alone. The mutant subunits KCNE2-

M23L or KCNE1-I57T increased current amplitudes even more, implying a gain-of-function, albeit not reaching statistical significance. We also investigated the effect of KCNE2 on steady-state inactivation (Figure 6C) and time-dependent recovery from inacti-vation (Figure 6D), yet we found no changes in these kinetic parameters.

Effect on the ultrarapid delayed rectifier currentWe furthermore investigated the effect of KCNE2 on the ultrarapid delayed rectifier current that is spe-cifically expressed in the atria. The ultrarapid delayed rectifier current is carried by the pore-forming subunit K

V1.5 that has been reported to interact with KCNE2

in mice [36]. We expressed KV1.5 alone or in the pres-

ence of KCNE2, KCNE2-M23L or KCNE2-I57T in X. laevis oocytes. However, we detected no differ-ences neither with respect to current amplitudes or biophysical parameters (data not shown).

DiscussionIn this study, we identified two missense mutations (M23L and I57T) in potassium channel β-subunit KCNE2 [37] as possible genetic substrates for early-onset lone AF.

In an attempt to characterize these mutations func-tionally, we coexpressed them with potassium chan-nel α-subunits that have previously been described to interact with KCNE2, namely K

V7.1, K

V11.1, K

V4.3

(4.2) and KV1.5 in a heterologous expression system.

To control expression levels and subunit ratios we used the X. laevis expression system, which allows for direct injection of precise cRNA amounts.

Coexpression of KCNE2-WT with KV7.1 generated

currents with decreased current density compared with K

V7.1 expressed alone. This current reducing effect of

KCNE2 was lost for the mutant KCNE2 β-subunits as coexpression of the mutant β-subunits with K

V7.1





Figure 5. Effect of KCNE2 wild-type or KCNE2 mutants on the rapid delayed rectifier current. (A) Representative current traces recorded at test potentials from -60 mV to +40 mV from Xenopus laevis oocytes expressing KV11.1 alone (left) or when coexpressed with wild-type KCNE2 (right), KCNE2-M23L or KCNE1-I57T. Currents were elicited using the voltage-clamp protocol shown in the inset to construct current–voltage relationships. (B) Steady-state currents measured at the end of each step, were normalized to the maximum current measured from X. laevis oocytes expressing KV11.1 alone, and plotted as a function of the test voltage. (C) Voltage dependence of activation was investigated by plotting the normalized peak tail current measured at -60 mV against the preceding step potential. The normalized data were fit to a two-state Boltzmann distribution. (D) To investigate the voltage-dependent release from inactivation, currents were elicited by the voltage protocol shown in the inset. Normalized peak currents measured at +40 mV were plotted against the preceding step potential and fitted to a two-state Boltzmann distribution. (KV11.1, KV11.1+KCNE2, KV11.1+KCNE2-M23L and KV11.1+KCNE2- I57T: n = 26, 20, 23 and 15, respectively). I/IMax: Current divided by maximal current; Vm: Membrane potential.

Kv11.1 Kv11.1+KCNE2

Kv11.1

Kv11.1+KCNE2

Kv11.1+KCNE2-I57L

Kv11.1+KCNE2-M23L

1 s

5 µA

40 mV

-60 mV-80 mV

2 s

0.0

0.5

1.0

0.0

0.0

0.5

0.5

1.0

1.0

1.5

No

rmal

ized

cu

rren

t

Vm (mV)

Vm (mV)

Vm (mV)

-40 -20 0 20 40

-40 -20 0 20 40

I Tai

l/IT

ail m

axI/I

max

0-20-40-60-80-100-120-140-160

40 mV

2 s

-160 mV5 ms


led to a significant increase of potassium current, hence a gain-of-function, both regarding peak current amplitudes and activation kinetics, compared with coexpression of K

V7.1 and KCNE2-WT. This increase

in current amplitude was not due to changes in sub-cellular localization of the β-subunits as confocal imaging did not reveal changes in localization of WT

or mutant KCNE2. In mice, the KCNE2-KV7.1 chan-

nel complex is essential for normal thyroid hormone function and both KCNE2 and K

V7.1 are expressed

in human thyroid tissue [38]. It could, thus, be specu-lated that the present KCNE2 mutations could cause AF through an intermediate pheno type of altered thy-roid function, as hyperthyroidism disposes to AF [38].



Figure 6. Effect of KCNE2 wild-type or KCNE2 mutants on the transient outward current. (A) Representative current traces recorded at test potentials from -60 mV to +40 mV from Xenopus laevis oocytes expressing KV4.3+KChIP2 alone (left) or when coexpressed with wild-type KCNE2 (right), KCNE2-M23L or KCNE1-I57T. Currents were elicited using the voltage-clamp protocol shown in the inset to construct current–voltage relationships. (B) Peak currents were normalized to the maximum current measured from X. laevis oocytes expressing KV4.3+KChIP2 alone, and plotted as a function of the test voltage. Two-way analysis of variance statistical analyses followed by a Bonferroni post-test were performed to compare the effect of the KCNE2 mutant on the current/voltage relationship: KV4.3+KChIP2 versus KV4.3+KChIP2+KCNE2 (†); KV4.3+KChIP2 versus KV4.3+KChIP2 +KCNE2-M23L (‡); KV4.3+KChIP2 versus KV4.3+KChIP2 +KCNE2-I57T (§). (C) To investigate the effect of KCNE2 on steady-state inactivation, currents were elicited by the voltage protocol shown in the inset. Normalized peak currents measured at +10 mV were plotted against the prepulse potential and fitted to a two-state Boltzmann distribution. (D) To investigate the time-dependent recovery from inactivation, a two-pulse protocol with increasing interpulse intervals was used (see inset). The peak current at the second pulse was normalized to the current at the first pulse and a single exponential equation was fitted to the data. (KV4.3+KChIP2, KV4.3+KChIP2+KCNE2, KV4.3+KChIP2+KCNE2-M23L, and KV4.3+KChIP2+KCNE2- I57T: n = 11, 15, 16, and 12, respectively). I/IMax: Current divided by maximal current; Vm: Membrane potential.

A B

C D

500 ms

5 µA

KV4.3+KChIP2 KV4.3+KChIP2+KCNE2

-60 -40 -20 0 20 40

0

1

2

Vm (mV)

Vm (mV)

No

rmal

ized

cu

rren

t

-120 -100 -80 -600.0

0.5

1.0

I/Im

ax

I/Im

ax

0 20 40 60 80 100 120 140 160 180 2000.0

0.5

1.0

Time (ms)

-80 mV

-80 mV

-80 mV

40 mV

1 s

10 mV

2 s

10 mV

0–200 ms

-120 mV

500 ms

KV4.3+KChIP2

KV4.3+KChIP2+KCNE2

KV4.3+KChIP2+KCNE2-M23L

KV4.3+KChIP2+KCNE2-I57T

†††

§§§‡‡‡

††

§§§‡‡‡

†

§§§‡‡‡

‡‡§§§

‡§


However, we did not observe thyroid disturbances in any of the two probands.

Noteworthy, the effect of KCNE2 mutations on K

V7.1 was abolished in the presence of the major I

Ks

β-subunit KCNE1, when coexpressed in a 1:1:1 molar ratio. Other studies have suggested that saturating amounts of KCNEs are necessary for full effects of the auxiliary subunits on K

V7.1 [27,39]. Indeed, coexpressing

KV7.1, KCNE1 and KCNE2 in a 1:4:4 ratio revealed

a significant gain-of-function of the mutated channel complexes as compared with when WT KCNE2 was coexpressed. The increased steady-state current and hyperpolarized shift (∼10 mV) in channel activation would suggest an increase in I

Ks current, resulting in

a shortened cardiac action potential. Shortened atrial action potential is a hallmark of the electrical remod-eling that takes place in AF. In particular, shorten-ing of the action potential duration will abbreviate refractoriness and wave length, and thereby facilitate re-entry arrhythmia [40]. Of note, also K

V11.1 and

KV4.3/KChIP2 coexpressed with the mutants showed

a tendency to higher current densities compared with WT KCNE2, albeit not reaching statistical signifi-cance. Hence, the phenotype seen in the patients may be due to a composite effect of the KCNE2 mutations.

Further support for a more complex pattern comes from previous studies that have associated mutations in KCNE2 with both drug-induced and acquired long QT syndrome (LQTS), Brugada syndrome (BrS) type 1 ECG phenotypes [35] and also AF (KCNE2 R27C) [10]. Abbott and Goldstein et al. reported four missense mutations (Q9E, M54T, I57T and A116V) in patients with different LQTS phenotypes including the muta-tion I57T, which we also found in one of our lone AF subjects. All four mutations, including I57T, led to a K

V11.1-KCNE2 loss-of-function, mainly through

increased voltage dependence of channel activation [41,42]. We did not observe this suppressing effect of KCNE2 on K

V11.1 channel function, yet, different

expression systems were used in the two studies. The mutation I57T has additionally been reported in three unrelated patients with BrS type 1 ECG phenotypes [35]. The authors demonstrated that KCNE2 associ-ates with K

V4.3 and KChIP2b, thereby contributing to

Ito. KCNE2 I57T resulted in I

to gain-of-function. The

authors speculated that this increased Ito conductance

could result in loss of the epicardial action potential dome during phase 1 of the action potential, thus pro-moting a substrate for phase 2 re-entry and risk of fatal ventricular arrhythmias [35]. Of note, we also found an increase of I

to conductance for both KCNE2 mutants,

yet not reaching significance.Neither of our two lone AF probands, including

the patient with the mutation I57T, had evidence of

LQTS and/or BrS, as neither of the patients had a history of syncope or near syncope, as there was no family history of sudden unexpected death, and as the sinus rhythm ECGs had no evidence of prolonged QTc intervals or BrS ECG patterns.

With the present finding of KCNE2 I57T in a lone AF patient, the same mutation has then been reported in three different arrhythmic diseases. This finding is rather perplexing as the prevailing conceptual model of LQTS with enhanced electrical refractoriness as a substrate for arrhythmias is opposite to the most common theory of AF genesis and to some extent also to the theory of BrS, where mainly reduced electrical refractoriness is considered as substrate of arrhyth-mia. However, it has previously been described that a single SCN5A mutation can cause both LQTS and BrS within the same family [43]. In a cohort of con-genital LQTS patients, Ackermann and colleagues reported a relatively high prevalence of early-onset lone AF [44], and in a Chinese family, nine of 16 family members diagnosed with autosomal domi-nant hereditary AF showed prolonged QTc ranging from 450 to 530 ms [7]. These findings, together with the present results, indicate that the same mutation might predispose to different arrhythmogenic phe-notypes. Additionally, we have recently shown that both short and long QTc intervals are associated with an increased risk of incident AF, indicating that both reduced and enhanced cardiac repolarization might predispose to AF [45]. Finally, the fact that both genetic variants associated with a slower and variants associated with a faster heart rate confer an increased risk of AF indicates that the pathophysiology under-lying AF is more complex than can be explained by a few simplified models [46].

To our knowledge, the KCNE2 mutation M23L is a novel finding as the mutation has not previously been reported in the literature, in publicly available databases, in 1500 alleles from our own control pop-ulation, or in 13,006 alleles on the NHLBI Exome Sequencing Project Exome Variant Server. This sup-ports the significance of M23L. The mutation I57T was absent in our control population of individuals free of heart diseases but was present in ESP with very low frequency (two out of 8600 [0.02%] alleles from individuals of European–American ethnicity). This indicates that KCNE2 I57T, if disease causing at all, is more likely a susceptibility variant rather than a monogenic cause of AF. However, the fact that the mutation has been reported in several patients with various arrhythmic syndromes and now AF, and the fact that the mutation has a functional effect, indicates that the mutation might be a substrate for cardiac arrhythmia in general.




Screening of KCNE3 revealed the variant R83H in one lone AF patient and in one healthy control person. Despite the fact that KCNE3 R83H has been reported in association with familial periodic paraly-sis and despite the fact that the variant has electro-physiological importance [47], this variant has most likely none or only very little effect on arrhythmic disease as the variant is present in ESP with an allele frequency of 0.7% and as the variant additionally has been reported in 1% of healthy individuals [48].

Study limitationsAs is the case for most other studies applying the can-didate gene sequencing approach, we cannot rule out that the identified mutations are part of the genetic diversity and without impact on disease. Addition-ally, neither of our two lone AF probands with muta-tions in KCNE2 had a family history of AF. How-ever, AF is only in extremely rare cases considered a monogenic disease; our study was a replication of a previous report also indicating that gain-of-function mutations in KCNE2 might underlie AF [10]; and both our functional studies and bioinformatic inves-tigations points towards that the identified KCNE2 mutations might play a role in AF.

We limited our analysis to the coding regions of KCNE2 and KCNE3, and it is possible that genetic variation in other regions of these genes could pose a substrate for AF. The functional analyses used con-ventional heterologous expression systems; hence, the environments differ from that in the native cardiomyocyte.

ConclusionIn this study of 192 early-onset lone AF patients, we identified two suspected disease-modifying muta-tions in KCNE2, both leading to significant gain-function when coexpressed with the K

V7.1 channel.

KCNE3 was not associated with lone AF in our study.

Future perspectiveDuring previous years we have witnessed a plethora of innovative and important findings of rare muta-tions in cardiac ion channel genes that could mecha-nistically explain the patient’s phenotype. At the same time, increasing evidence points to more com-plex scenarios, for example the same mutation can cause different phenotypes in different individuals even within one family, the effect of a mutation or rare variant can be modified by common variants, or mutations in one ion channel subunit may affect several ionic currents due to a crosstalk of subunits, to mention a few. This complex pattern will chal-lenge our methodological way of investigating the functional impact of rare and common variants. To study the consequences of the genetic variants, we believe that the use of patient-specific induced plu-ripotent stem cell derived cardiomyocytes will be pivotal. Compared to the exogenous ion channels expression in frog eggs or mammalian cell lines, the consequences of the genetic variants can be tested in a more relevant model environment – that is, cardio-myocytes derived from the patients themselves. Such work could aid in discovering the cellular mecha-nism behind AF and help in the development of more personalized medicine.

AcknowledgementsThe authors are grateful to all participating patients.

Financial & competing interests disclosureThis work was financially supported by The Danish National

Research Foundation; The John and Birthe Meyer Founda-

tion; Landsforeningen til Bekæmpelse af Kredsløbssygdom-

me; Lægernes Forsikringsforening af 1891; Kong Christian

Den X’s Fond; Margrethe Møller Fonden; The Arvid Nilsson

Foundation; Director Ib Henriksens Foundation; Fonds-

børsvekselerer Henry Hansen og Hustru Karla Hansen, født

Westergaards Legat; and The Danish Heart Foundation.



Executive summary

Mutation screening & bioinformatics reveal possible disease-causing variants in a lone atrial fibrillation population• Diseases resulting from compromised ion channel function, channelopathies, are becoming increasingly

recognized in human pathology and cardiovascular disease.• Two possibly disease-modifying gain-of-function missense mutations in KCNE2 associated with early-onset

lone atrial fibrillation were identified. KCNE3 was not associated with lone atrial fibrillation in our study.Functional characterization of the KCNE2 mutations shows a composite effect of the mutations on several ionic currents• Coexpression of mutant β-subunits with KV7.1 led to a significant gain-of-function in current amplitudes.• Coexpression with saturating amounts of KCNEs resulted in a significant gain-of-function of the cardiac slow

delayed rectifier current.• Changes in subcellular localization could not explain the increased current amplitudes found for the β-subunit

mutants.


The authors have no other relevant affiliations or financial

involvement with any organization or entity with a finan-

cial interest in or financial conflict with the subject matter

or materials discussed in the manuscript apart from those

disclosed.

No writing assistance was utilized in the production of this

manuscript.

Ethical conduct of researchThe authors state that they have obtained appropriate institu-

tional review board approval or have followed the principles

outlined in the Declaration of Helsinki for all human or animal

experimental investigations. In addition, for investigations in-

volving human subjects, informed consent has been obtained

from the participants involved.

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