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Research Article For reprint orders, please contact: [email protected]
Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation
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 (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 plasmids 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 & imaging
Transiently transfected CHO-K1 cells grown on glass coverslips were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min at room temperature. 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, Denmark). 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 (Martinsried, 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
Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation
Research Article
anti-K V7.1 (2 μg/ml, C-20; Santa Cruz Biotechnology, 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 donkey anti-goat IgG (4 μg/ml), Alexa-Fluor 647 phalloidin (1.5 U/ml), rhodamine-conjugated phalloidin (1.5 U/ml). All Alexa Fluor®-coupled reagents and phalloidin were from Invitrogen (Glostrup, Denmark).
Results
Two-electrode voltage clamp
Mutation screening & bioinformatics
Xenopus 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 (K V7.1: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 DMZUniversal Puller (Zeitz Instruments, Martinsried, Germany) 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, MgCl2 1 mm, CaCl2 1 mm, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 5 mm, pH = 7.4 with NaOH) and experiments 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.
Direct DNA sequencing of KCNE2 in 192 index patients of Danish/Caucasian ethnicity revealed two nonsynonymous mutations: c.67A>T, leading 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 previously 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 functional importance (Figure 1) . Both M23L and I57T
Study cohort
The study population consisted of 192 patients with onset of AF ranging from 16 to 39 years, without any concomitant disease. The two control populations 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.
Table 1. Clinical characteristics of the lone atrial fibrillation population (n = 192).
Data analysis
Parameter
Value
Data analysis was performed with Igor Pro (Wavemetrics, OR, USA) and GraphPad Prism (GraphPad Software Inc., CA, USA). A Boltzmann function:
Median age of onset; years (IQR)
31.5 (26–36)
Male gender (%)
82
Height (cm)
183 ± 9
Weight (kg)
89 ± 17
BMI (kg/m )
26.7 ± 4.6
2
Blood pressure (mmHg)
was fitted to activation and inactivation curves to obtain the potential of half-maximal activation/inactivation (V50) and the slope factor (k). Data are represented as mean ± standard error of the mean, unless otherwise indicated. Two-way analysis of variance statistical analyses followed by a Bonferroni post-test or analysis of variance followed by Tukey’s method of multiple comparisons were used as appropriate to compare the wild-type and mutated KCNE2 channel complex. A two-sided p-value A, leading to an exchange of arginine 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 data
Proband 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 0.05]; K V7.1+KCNE2 vs K V7.1+KCNE2-M23L [p 0.05]; K V 7.1+KCNE1+KCNE2 versus K V 7.1+KCNE1+KCNE2-M23L [p
Research Article For reprint orders, please contact: [email protected]
Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation
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 (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 plasmids 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 & imaging
Transiently transfected CHO-K1 cells grown on glass coverslips were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 15 min at room temperature. 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, Denmark). 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 (Martinsried, 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
Gain-of-function mutations in potassium channel subunit KCNE2 associated with early-onset lone atrial fibrillation
Research Article
anti-K V7.1 (2 μg/ml, C-20; Santa Cruz Biotechnology, 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 donkey anti-goat IgG (4 μg/ml), Alexa-Fluor 647 phalloidin (1.5 U/ml), rhodamine-conjugated phalloidin (1.5 U/ml). All Alexa Fluor®-coupled reagents and phalloidin were from Invitrogen (Glostrup, Denmark).
Results
Two-electrode voltage clamp
Mutation screening & bioinformatics
Xenopus 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 (K V7.1: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 DMZUniversal Puller (Zeitz Instruments, Martinsried, Germany) 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, MgCl2 1 mm, CaCl2 1 mm, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 5 mm, pH = 7.4 with NaOH) and experiments 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.
Direct DNA sequencing of KCNE2 in 192 index patients of Danish/Caucasian ethnicity revealed two nonsynonymous mutations: c.67A>T, leading 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 previously 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 functional importance (Figure 1) . Both M23L and I57T
Study cohort
The study population consisted of 192 patients with onset of AF ranging from 16 to 39 years, without any concomitant disease. The two control populations 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.
Table 1. Clinical characteristics of the lone atrial fibrillation population (n = 192).
Data analysis
Parameter
Value
Data analysis was performed with Igor Pro (Wavemetrics, OR, USA) and GraphPad Prism (GraphPad Software Inc., CA, USA). A Boltzmann function:
Median age of onset; years (IQR)
31.5 (26–36)
Male gender (%)
82
Height (cm)
183 ± 9
Weight (kg)
89 ± 17
BMI (kg/m )
26.7 ± 4.6
2
Blood pressure (mmHg)
was fitted to activation and inactivation curves to obtain the potential of half-maximal activation/inactivation (V50) and the slope factor (k). Data are represented as mean ± standard error of the mean, unless otherwise indicated. Two-way analysis of variance statistical analyses followed by a Bonferroni post-test or analysis of variance followed by Tukey’s method of multiple comparisons were used as appropriate to compare the wild-type and mutated KCNE2 channel complex. A two-sided p-value A, leading to an exchange of arginine 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 data
Proband 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 0.05]; K V7.1+KCNE2 vs K V7.1+KCNE2-M23L [p 0.05]; K V 7.1+KCNE1+KCNE2 versus K V 7.1+KCNE1+KCNE2-M23L [p
