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A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation
BBRC Biochemical and Biophysical Research Communications 332 (2005) 1012–1019 www.elsevier.com/locate/ybbrc
A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation Min Xia a,1, Qingfeng Jin a,b,1, Saı¨d Bendahhou c,1, Yusong He a,b,1, Marie-Madeleine Larroque c, Yiping Chen d, Qinshu Zhou a, Yiqing Yang b, Yi Liu a, Ban Liu a, Qian Zhu b, Yanting Zhou d, Jie Lin b, Bo Liang a, Li Li a, Xiongjian Dong b, Zhiwen Pan a, Rongrong Wang b, Haiying Wan b, Weiqin Qiu a, Wenyuan Xu b, Petra Eurlings a, Jacques Barhanin c, Yihan Chen a,b,* a Institute of Medical Genetics, Tongji University, Shanghai, China Department of Cardiology, Tongji Hospital, Tongji University, Shanghai, China c Institut de Pharmacologie Mole´culaire et Cellulaire, UMR 6097 CNRS, Valbonne, France Department of Cardiology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH, USA b
d
Received 4 May 2005 Available online 23 May 2005
Abstract The inward rectifier K+ channel Kir2.1 mediates the potassium IK1 current in the heart. It is encoded by KCNJ2 gene that has been linked to AndersenÕs syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the lossof-function effect of previously reported mutations in AndersenÕs syndrome. Kir2.1 V93I mutation may play a role in initiating and/ or maintaining AF by increasing the activity of the inward rectifier K+ channel. 2005 Elsevier Inc. All rights reserved. Keywords: Kir2.1; KCNJ2; Ion channel; Atrial fibrillation; Molecular genetics
As the most common cardiac arrhythmia in clinical practice, atrial fibrillation (AF) is characterized by rapid and irregular activation of the atrium. AF prevalence increases progressively with age, with nearly 6% in those over 65 years of age. A recent Framingham Heart Study showed that lifetime risks for AF development were about 25% for men and women 40 years of age and older. AF may cause strokes and heart failures, and is a source of considerable morbidity and mortality [1–4]. However, to date, a definitive treatment approach has *
1
Corresponding author. Fax: +86 21 66371663. E-mail address: [email protected] (Y. Chen). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.054
not been established. According to a large survey performed in the arrhythmia clinic at Mayo Clinic in Rochester, Minnesota, about 36% patients with AF had no evident pathogeny, and 5% had a positive family history [5]. This points to the fact that a subset of AF has an important genetic background. Recently, we found two causative genes (K+ channel genes KCNQ1 and KCNE2) for familial AF [6,7]. However, more molecular basis or causative genes remain to be identified [5–10]. Understanding the electrophysiological process leading to AF has been a subject of intensive study over last century. Dobrev et al. [11,12] observed that the current density of IK1 in human atrial myocytes was twofold
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
greater during AF than in sinus rhythm. Van Wagoner et al. [13,14] and Bosch et al. [15] also noted a significant increase in IK1 in atrial myocytes of patients with AF. Kir2.1 to Kir2.4 subfamily of inward rectifier K+ channel family underlies cardiac IK1 [16,17]. Kir2.1 is encoded by KCNJ2, a causative gene of Andersen syndrome, which is characterized by a triad of periodic paralysis, cardiac arrhythmia, and skeletal developmental abnormalities [18]. The role of Kir2.1 in forming cardiac IK1 was supported by the absence of IK1 current in cardiac myocytes from Kir2.1/ mice [19]. The overexpression of Kir2.1 in the mouse heart upregulated IK1 and ultimately initiated multiple abnormalities of cardiac excitability including AF [20]. In this study, we analyzed the Kir2.1 coding gene KCNJ2, the congenital long QT syndrome-associated genes (KCNQ1, HERG, SCN5A ANK-B, KCNE1, and KCNE2), and several other channel genes (KCNE3, KCNE4, and KCNE5) in 30 unrelated Chinese kindreds with AF. We found a missense mutation in KCNJ2 in one Chinese family. Heterologous expression in COS and HEK cells revealed a gain-of-function effect consistent with the AF clinical manifestations seen in this family.
Materials and methods Subjects. We identified 30 unrelated kindreds of familial AF among Chinese Han population. The diagnostic criteria for familial AF were a positive AF family history, electrocardiographic evidence of AF, and exclusion of organic heart diseases and other causative factors. We screened 10 ion channel or transporter related genes (KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5) (GenBank Accession Nos. AJ006345, NT_007914, NT_022517, NW_105991, AP001720, NM_005136, NT_035430, XM_208561, NT_005403, and NM_012282. GenBank: http:// www.ncbi.nlm.nih.gov/GenBank) in these 30 probands and in their family members. The controls were 420 unrelated healthy Chinese subjects of Han nationality. The study was approved by the Ethical Committee of Chinese Human Genome Center at Shanghai. Written informed consent was obtained from all subjects. Genetic analysis. DNA was extracted from whole venous blood with Wizard Genomic DNA Purification Kit (Promega). All coding sequences of the KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5 were amplified using HotStarTaq DNA Polymerase (Qiagen). Products of PCR amplification were purified with MBI Fermentas DNA Extraction Kit (MBI). Both strands of each PCR product were sequenced with a DYEnamic ET dye terminator kit (Amersham Biosciences) under MegaBACE 500 DNA Sequencing system (Amersham Biosciences). RT-PCR. For RT-PCR, human atrial and ventricular tissue specimens were collected and preserved in RNAlater RNA stabilization reagent (Qiagen). Total RNA was prepared using an RNeasy Protect Mini kit (Qiagen). An on-column DNase treatment was included in the RNA isolation step. Before RT-PCR, RNA samples were further treated with DNase (Invitrogen). RT was performed with KCNJ2 (5 0 GTGACACATCTGAAACCATAGCC-3 0 ) and control gene GAPDH (5 0 -CCACCACCCTGTTGCTGTAG-3 0 ) specific primers using SuperScriptIIreverse transcriptase (Invitrogen). PCR was performed in a 25 ll reaction mixture with an annealing temperature at 62 C, and
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35 cycles. Q-solution was included in the reactions for the KCNJ2 and GAPDH transcripts. The primer pairs used for the concurrent amplification of the two transcripts were for KCNJ2: forward—5 0 TCAGTAGACAGACCTTGGTAGAACC-3 0 , reverse—5 0 -GTGA CACATCTGAAACCATAGCC-3 0 , product size 621 bp; for GAPDH: forward—5 0 -GCCAAGGTCATCCATGACA-3 0 , reverse—5 0 -CCAC CACCCTGTTGCTGTAG-3 0 , product size 496 bp. Genomic DNAnegative control for PCR was conducted by adding RNA template instead of RT product to the PCR mixture. Mutagenesis. Forward (5 0 -GCAGGATCCAAAGCAGAAGCA CTGGAGTC-3 0 ) and reverse (5 0 -GCAGCGGCCGCTGACCCAT CTTGACCAGTACC-3 0 ) primers were used to amplify the coding regions and untranslated flanking regions of the human KCNJ2 gene using pfuUltra high-fidelity DNA polymerase (Stratagene). Purified, BamHI and NotI digested PCR product was subcloned into the BamHI and NotI sites of the pXOOM expression vector (kindly provided by Dr. T Jespersen, University of Copenhagen, Denmark). Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutageneses Kit. The forward and reverse primers were 5 0 -ATCTTCTGCCTGGCTTTCATCCTGTCATGGCTGTT TT-3 0 and 5 0 -AAAACAGCCATGACAGGATGAAAGCCAGGCA GAAGAT-3 0 , respectively. The entire coding region of the mutant clone was confirmed by sequencing. Transfection and electrophysiology. Cells (COS-7 or HEK293) were maintained as described [6]. Cells were transiently transfected by DEAE–dextran precipitate method with 2 lg wild-type (WT) KCNJ2pXOOM DNA or 2 lg mutant-type (MT) KCNJ2-pXOOM DNA or 1 lg WT and 1 lg MT KCNJ2-pXOOM DNA per 60-mm culture dish. Current recordings were performed 48 h after transfection in the whole-cell configuration at room temperature (22 C), using EPC-10 amplifier (HEKA Instruments), and as previously described [6]. Confocal microscopy. In order to monitor Kir2.1 subcellular localization, WT KCNJ2 and MT KCNJ2 were transferred into the pEYFP-N1 vector (enhanced yellow fluorescent protein, Clontech) and the pECFP-N1 vector (enhanced cyan fluorescent protein, Clontech), respectively. The whole coding region of the two plasmid constructs was confirmed by sequencing. Fluorescence microscopy was performed with a Leica confocal microscopy system. Statistical analysis. Current changes were assessed by means of ANOVA and DunnettÕs t test. All reported p values are two-sided and a p value of 0.05 or less was considered to indicate statistical significance. Data are given as means ± SE.
Results Identification of Kir2.1 V93I mutation The probands in 30 kindreds of familial AF were clinically and genetically evaluated. None of them had evident etiologies for AF. We sequenced 10 ion channel or transporter related genes (KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5) after PCR amplification of genomic DNA. A novel missense mutation in KCNJ2 was found in one of the probands. With several DNA samples unavailable and the phenotypes of II:12, III:3, and III:6 uncertain, using the flanking microsatellite D17S949, a two-point linkage analysis between KCNJ2 and the familial AF gave a LOD score of 1.93 at recombination fraction 0 (detailed data not shown). The proband (II-3) who had paroxysmal AF
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as frequently as two to three times a month was a heterozygote for the 277G fi A transition, corresponding to a V93I substitution in KCNJ2. His father (I-1) and older sister (II-2) had permanent AF before death. V93I was also found in his younger sister (II-6), niece (III-2), and nephews (III-3 and III6). His younger sister (II-6) experienced paroxysmal AF as frequently as once or twice a week. His niece (III-2) showed paroxysmal AF on 24-h electrocardiographic recordings. His nephews (III-3 and III-6 who was 42 and 33 years old, respectively) did not have AF on 24-h electrocardiographic monitoring. In view of the characteristic of paroxysmal onset of AF, we did not exclude that III3 and III-6 were AF patients. We did not obtain the blood sample of II-12 and did not know her phenotype. Other unaffected family members did not carry the V93I substitution. The substitution co-segregated with AF in the family with the exception of III-3 and III-6 (Table 1 and Fig. 1). All affected members in this family had normal QT interval on 12-lead ECG, experienced no episodes of muscle weakness or syncope, and did not have frequent premature ventricular contractions or ventricular tachycardia on 24-h electrocardiographic monitoring. Serum potassium levels were within normal limits. None of the patients exhibited any developmental problems such as cleft palate, low set ears, short stature, clinodactyly, syndactyly, or brachydactyly, as reported for AndersenÕs syndrome. To further confirm that the V93I substitution of Kir2.1 was not a benign polymorphism, we checked this substitution in 420 healthy individuals. It was not found in any of these healthy individuals. Kir2.1 expression in human atrium Although Kir2.1 is expressed in human heart, its distribution in different heart chambers is not clear. Qualitative RT-PCR was performed to determine the tissue distribution of KCNJ2 mRNA in the human heart. Our data showed that Kir2.1 was strongly expressed in both ventricles and atria, with lower signals in atria (Fig. 1).
Conservation of sequences flanking Kir2.1 V93I mutation Kir2.1 subunits are made of two membrane-spanning segments (M1 and M2) adjacent to a pore-forming loop, and cytoplasmic N- and C-termini. V93I substitution is located in the outer helix of the M1 domain [21] which displays a highly conserved sequence among human, domestic guinea pig, pig, dog, cow, Norway rat, rabbit, and house mouse (Table 2 and Fig. 2). V93I substitution perturbs a highly conserved residue suggesting that V93I may have important functional consequences. Functional effect of V93I mutation on Kir2.1 channel We expressed WT and MT KCNJ2 in COS-7 cells, and performed electrophysiological study using wholecell patch-clamping technique. The data were as follows: (1) the currents for MT expression at potentials ranging from 140 to 80 mV or from 60 to 40 mV had amplitudes significantly higher than those for WT expression. For example, at 90 mV, the current for MT expression was 97 ± 7.8 pA/pF (n = 20) and that for WT expression 40 ± 4.9 pA/pF (n = 22) (p < 0.01); whereas at 50 mV, the former was 97 ± 8.0 pA/pF (n = 20) and the latter 15 ± 3.0 pA/pF (n = 22) (p < 0.001) (Fig. 3); (2) at the same potential range, co-expression of MT and WT also significantly enhanced the Kir2.1 current (Fig. 3); (3) compared with the currents for co-expression of MT and WT, the currents for MT expression at potentials ranging from 50 to 40 mV were significantly larger (Fig. 3). Similar results were obtained in HEK293 cells (data not shown). Hence, it is concluded that the V93I mutation had a gain-of-function effect on Kir2.1 channels. Subcellular trafficking of mutant Kir2.1 channel To assess the subcellular trafficking of WT and MT KCNJ2 subunits, we performed confocal laser scanning microscopy in COS-7 and HEK293 cells. Cells transfected with WT KCNJ2 showed the same plasma membrane
Table 1 Clinical characteristics of subjects with Kir2.1 V93I mutation Family member
Sex
Age (year)
Age at AF diagnosis (year)
Recurrent palpitation
AF classification
Ventricular rate (bpm)
QTc (s)
LAD (mm)
LVEF (%)
I-1 II-2 II-3 II-6 III-2 III-3 III-6
M F M F F M M
82a 56a 59b 56b 57b 42b 33b
58 50 54 50 57 NA NA
+ + + +
Permanent Permanent Paroxysmal Paroxysmal Paroxysmal NA NA
NA 63/70c 68/110c 71/118c 66/102c 83 66
NA 0.42/0.43c 0.41/0.44c 0.43/0.43c 0.42/0.43c 0.41 0.37
NA 35 36 35 31 33 34
NA 73 65 66 71 68 65
QTc, corrected QT interval; NA, not available or not applicable; LAD, left atrial dimension; LVEF, left ventricular ejection fraction. a The age at death is shown. b Age is the current age. c The value in AF is shown.
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
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Fig. 1. Familial AF associated with Kir2.1 V93I mutation. (A) Pedigree for a Chinese AF kindred. Asterisks indicate that the individual had the V93I mutation. (B) Normal KCNJ2 DNA sequence. (C) KCNJ2 DNA sequence of the affected family members. Sequence analysis of DNA from the affected family members after PCR amplification revealed a G-to-A substitution at nucleotide 277 in KCNJ2 causing a V93I mutation. (D) RT-PCR results for transcripts of KCNJ2 and GAPDH genes concurrently expressed in human atrial and ventricular tissues.
fluorescence pattern as those transfected with MT KCNJ2. In cells co-transfected with WT and MT KCNJ2, both proteins also demonstrated a similar fluorescence
pattern (data not shown). Tagging with the fluorescent protein did not affect the Kir2.1 channel function (data not shown).
Table 2 Conservation of sequences flanking Kir2.1 V93I mutation Species
Amino acids flanking residue 93
Identitiesa (%)
Human Mutant V931 Domestic guinea pig Pig Dog Cow Norway rat Rabbit House mouse
KGQRYLADIFTTCVDIRWRWMLVIFCLAFVLSWLFFGCVFWLIALLHGDLDASK ...............................................................................I............................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... .............................................................................................................................................R ..........................................................................................................................................T...
99 99 99 99 99 99 98
a
Identity is the percentage of the sequence that is identical to human sequence as based on protein sequences from the following GenBank ID (gi): Human Kir2.1, gi: 4504835; domestic guinea pig Kir2.1, gi: 1708549; pig Kir2.1, gi: 3024031; dog Kir2.1, gi: 13878561; Cow Kir2.1, gi: 27805969; Norway rat Kir2.1, gi: 2493597; rabbit Kir2.1, gi: 1352481; house mouse Kir2.1, gi: 547735.
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Fig. 2. The proximity of residue valine 93 to the Kir2.1 channel pore. Residue V93 on the Kir2.1 sequence aligns to residue V72 on the KirBac1.1 crystal structure and is located in the outer helix.
Discussion The availability of a large number of characterized AF kindreds in our cardiology center allowed us to identify KCNJ2 as a novel gene for familial AF. Interestingly, this gene has already been linked to AndersenÕs syndrome [22]. Unlike loss-of-function mutations in AndersenÕs syndrome, the V93I substitution identified in our AF family had a Kir2.1 gain-of-function effect. This mutation was present in all affected family members and absent in 420 healthy subjects. It was reported that overexpression of wild-type Kir2.1 in the mouse heart led to AF and other abnormalities of cardiac excitability [20]. Thus, we hypothesized that Kir2.1 mutations might be associated with familial AF. Before the Kir2.1 coding gene KCNJ2 was sequenced, we performed two-point linkage analysis on the four-generation family. Although several DNA samples were unavailable and the phenotypes of II:12, III:3, and III:6 uncertain, a LOD score of 1.93 was obtained at recombination fraction zero at D17S949 (data not shown). To date, over 20 Kir2.1 mutations have been found in families with AndersenÕs syndrome, all of which resulted in loss-of-function with dominant-negative suppression of inward currents, except for two, which displayed haplo-insufficiency [22–26]. In contrast, V93I had a gain-of-
function effect on Kir2.1 channels. The functional expression of the AF-associated V93I substitution demonstrated a significant gain-of-function in both inward and outward Kir2.1 currents, without affecting the kinetics and rectification properties. Although not quantified, the results with the fluorescently labeled Kir2.1 subunits are in favor of a normal trafficking of the mutated channels. A direct effect on channel conductance is more likely, as suggested by the proximity of residue valine 93 to the pore (Fig. 2). Kir2.1 plays a major role in both the late phase of repolarization (phase 3) and the phase of resting membrane potential (phase 4), but almost no current is allowed to pass the channel during the plateau phase of action potential [16,27–31]. At potentials negative to resting membrane potential, inward rectifier channels pass a large inward current to bring the potential back to the resting status. In contrast, at potentials positive to the resting membrane potential, inward rectifier channels pass a much smaller outward current to repolarize the cell. The activated inward rectifier channels may amplify and accelerate the rate of return of the membrane to its resting membrane potential value, leading to shortening of action potential duration [30,32,33]. As expected, the Kir2.1 loss-of-function mutations led to QT interval prolongation in AndersenÕs syndrome patients. Kir2.1 V93I mutation increased inward potassium current at 90 to 80 mV and outward potassium current at 60 to 40 mV. These would stabilize resting membrane potential and shorten the repolarization phase of the atrial action potential, resulting in a shortening of the atrial effective refractory period (ERP). Thus, the gain-of-function effect of V93I mutation may create a substrate favorable to a multiple wavelet re-entry, a dominant mechanism of AF [2]. An overexpression of Kir2.1 in mouse heart upregulated IK1 and initiated AF [20]. IK1 increase has also been reported in human acquired AF [11–14]. Additionally, all the two human AF-associated mutations reported to date increased K+ channel current [6,7]. The gain-of-function mutation of cardiac K+ channels could be a good trigger for AF. New evidence has been found to support the speculation. A gain-of-function mutation of HERG, a K+ channel, caused not only short QT syndrome but also AF [34]. According to the protein sequence alignment, the sequences flanking the KCNJ2 V93I substitution are well conserved in all mammals with KCNJ2 sequence known. However, the substitution can be found in birds (chickens and domestic pigeons) (GenBank Accession Nos.: chicken KCNJ2, gi: 45382445; domestic pigeon KCNJ2, gi: 6273353). This does not seem to support our hypothesis that KCNJ2 V93I is a novel mutation associated with familial AF. In view of this, we analyzed the expression of KCNJ2 in the hearts of chicken and domestic pigeon. We found that KCNJ2 was not
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
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Fig. 3. Functional expression of Kir2.1 V93I mutation in COS-7 cells. (A) Representative Kir2.1 currents. WT, currents from COS-7 cells transfected with wild Kir2.1 channels; WT + MT, currents from COS-7 cells co-transfected with wild and mutant Kir2.1 channels; MT, currents from COS-7 cells transfected with mutant Kir2.1 channels. (B) Kir2.1 current–voltage relationship. It was obtained from COS-7 cells expressing WT–WT Kir2.1 protein (j) (n = 22), WT–MT Kir2.1 protein (m) (n = 18), and MT–MT Kir2.1 protein (d) (n = 20). Currents were elicited by holding the cells to 80 mV, then cells were subjected to test pulses ranging from 140 to +50 mV for 200 ms. Values are means ± SE.
expressed in the atria of these two birds (data not shown). Thus, KCNJ2 may not participate in the physiological activity of the atrium in birds such as chicken and domestic pigeon, although it does in mammals. The appearance of the substitution in chicken and domestic pigeon does not conflict with the association of V93I with familial AF. Not all mutation carriers exhibited AF. The carriers (III-3 and III-6) did not have AF on a 24-h electrocardiographic monitoring, which may be explained by any or a combination of the following reasons: (1) these carriers were relatively young (33 and 42 years old) while AF usually occurs in aged patients; (2) paroxysmal AF occurs as rarely as a few times in life in some patients [3]. A longer duration of electrocardiographic monitoring may be required to record paroxysmal AF in these patients; (3) the familial AF caused by V93I mutation may have a low penetrance; (4) V93I may be only a genetic predisposing factor for AF. Environmental factors may also be involved in the onset of AF. We analyzed 154 patients with lone AF (data not shown). V93I mutation was not found in this popula-
tion, indicating that more linked genes remain to be identified in most of the AF patients without structural heart diseases. Kir2.1 gain-of-function mutation is one of the AF molecular bases. The study not only identifies a molecular mechanism of the genetic form of AF, but also sheds light on molecular mechanism of the acquired forms of AF. Kir2.1 channels or IK1 currents may be potential targets for drug therapy of AF.
Acknowledgments We are indebted to the family with AF for participating in the study. This work was supported by grants from the National Science Fund for Distinguished Young Scholars of China (30425016), the National Natural Science Foundation of China (30330290, 30170386, and 39900060), the Major Program Fund by the Ministry of Education of China (104081), the Shanghai Science and Technology Development Fund of China (SKW0202), the Shanghai Dawn Scholar Fund of China
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(SKW0103), and the Centre National de la Recherche Scientifique Fund of France.
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[16] A.N. Lopatin, C.G. Nichols, Inward rectifiers in the heart: an update on I(K1), J. Mol. Cell. Cardiol. 33 (2001) 625–638. [17] C. Zobel, H.C. Cho, T.T. Nguyen, R. Pekhletski, R.J. Diaz, G.J. Wilson, P.H. Backx, Molecular dissection of the inward rectifier potassium current (IK1) in rabbit cardiomyocytes: evidence for heteromeric coassembly of Kir2.1 and Kir2.2, J. Physiol. 550 (2003) 365–372. [18] E.D. Andersen, P.A. Krasilnikoff, H. Overvad, Intermittent muscular weakness, extrasystoles, and multiple developmental anomalies. A new syndrome? Acta Paediatr. Scand. 60 (1971) 559– 564. [19] J.J. Zaritsky, J.B. Redell, B.L. Tempel, T.L. Schwarz, The consequences of disrupting cardiac inwardly rectifying K+ current (IK1) as revealed by the targeted deletion of the murine Kir2.1 and Kir2.2 genes, J. Physiol. 553 (2001) 697–710. [20] J. Li, M. McLerie, A.N. Lopatin, Transgenic upregulation of IK1 in the mouse heart leads to multiple abnormalities of cardiac excitability, Am. J. Physiol. Heart Circ. Physiol. 287 (2004) H2790–H2802. [21] Y. Guo, G.J. Waldron, R. Murrell-Lagnado, A role for the middle C terminus of G-protein-activated inward rectifier potassium channels in regulating gating, J. Biol. Chem. 277 (2002) 48289–48294. [22] N.M. Plaster, R. Tawil, M. Tristani-Firouzi, S. Canun, S. Bendahhou, A. Tsunoda, M.R. Donaldson, S.T. Iannaccone, E. Brunt, R. Barohn, J. Clark, F. Deymeer, A.L. George Jr., F.A. Fish, A. Hahn, A. Nitu, C. Ozdemir, P. Serdaroglu, S.H. Subramony, G. Wolfe, Y.H. Fu, L.J. Ptacek, Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of AndersenÕs syndrome, Cell 105 (2001) 511–519. [23] S. Bendahhou, M.R. Donaldson, N.M. Plaster, M. TristaniFirouzi, Y.H. Fu, L.J. Ptacek, Defective potassium channel Kir2.1 trafficking underlies Andersen–Tawil syndrome, J. Biol. Chem. 278 (2003) 51779–51785. [24] M. Tristani-Firouzi, J.L. Jensen, M.R. Donaldson, V. Sansone, G. Meola, A. Hahn, S. Bendahhou, H. Kwiecinski, A. Fidzianska, N. Plaster, Y.H. Fu, L.J. Ptacek, R. Tawil, Functional and clinical characterization of KCNJ2 mutations associated with LQT7 (Andersen syndrome), J. Clin. Invest. 110 (2002) 381–388. [25] K. Ho, C.G. Nichols, W.J. Lederer, J. Lytton, P.M. Vassilev, M.V. Kanazirska, S.C. Hebert, Cloning and expression of an inwardly rectifying ATP-regulated potassium channel, Nature 362 (1993) 31–38. [26] H. Fodstad, H. Swan, M. Auberson, I. Gautschi, J. Loffing, L. Schild, K. Kontula, Loss-of-function mutations of the K(+) channel gene KCNJ2 constitute a rare cause of long QT syndrome, J. Mol. Cell. Cardiol. 37 (2004) 593–602. [27] Y. Shimoni, R.B. Clark, W.R. Giles, Role of an inwardly rectifying potassium current in rabbit ventricular action potential, J. Physiol. 448 (1992) 709–727. [28] Y. Kubo, T.J. Baldwin, Y.N. Jan, L.Y. Jan, Primary structure and functional expression of a mouse inward rectifier potassium channel, Nature 362 (1993) 127–133. [29] H.J. Jongsma, R. Wilders, Channelopathies: Kir2.1 mutations jeopardize many cell functions, Curr. Biol. 11 (2001) R747–R750. [30] J.M.B. Anumonwo, Biophysic properties of inward rectifier potassium channels, in: D.P. Zipes, J. Jalife (Eds.), Cardiac Electrophysiology: from Cell to Bedsides, first ed., Saunders, Philadelphia, 2004, pp. 112–119. [31] A.S. Dhamoon, S.V. Pandit, F. Sarmast, K.R. Parisian, P. Guha, Y. Li, S. Bagwe, S.M. Taffet, J.M. Anumonwo, Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current, Circ. Res. 94 (2004) 1332–1339. [32] B.A. Wible, M. De Biasi, K. Majumder, M. Taglialatela, A.M. Brown, Cloning and functional expression of an inwardly rectifying K+ channel from human atrium, Circ. Res. 76 (1995) 343– 350.
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A Kir2.1 gain-of-function mutation underlies familial atrial fibrillation Min Xia a,1, Qingfeng Jin a,b,1, Saı¨d Bendahhou c,1, Yusong He a,b,1, Marie-Madeleine Larroque c, Yiping Chen d, Qinshu Zhou a, Yiqing Yang b, Yi Liu a, Ban Liu a, Qian Zhu b, Yanting Zhou d, Jie Lin b, Bo Liang a, Li Li a, Xiongjian Dong b, Zhiwen Pan a, Rongrong Wang b, Haiying Wan b, Weiqin Qiu a, Wenyuan Xu b, Petra Eurlings a, Jacques Barhanin c, Yihan Chen a,b,* a Institute of Medical Genetics, Tongji University, Shanghai, China Department of Cardiology, Tongji Hospital, Tongji University, Shanghai, China c Institut de Pharmacologie Mole´culaire et Cellulaire, UMR 6097 CNRS, Valbonne, France Department of Cardiology, University Hospitals of Cleveland, Case Western Reserve University, Cleveland, OH, USA b
d
Received 4 May 2005 Available online 23 May 2005
Abstract The inward rectifier K+ channel Kir2.1 mediates the potassium IK1 current in the heart. It is encoded by KCNJ2 gene that has been linked to AndersenÕs syndrome. Recently, strong evidences showed that Kir2.1 channels were associated with mouse atrial fibrillation (AF), therefore we hypothesized that KCNJ2 was associated with familial AF. Thirty Chinese AF kindreds were evaluated for mutations in KCNJ2 gene. A valine-to-isoleucine mutation at position 93 (V93I) of Kir2.1 was found in all affected members in one kindred. This valine and its flanking sequence is highly conserved in Kir2.1 proteins among different species. Functional analysis of the V93I mutant demonstrated a gain-of-function consequence on the Kir2.1 current. This effect is opposed to the lossof-function effect of previously reported mutations in AndersenÕs syndrome. Kir2.1 V93I mutation may play a role in initiating and/ or maintaining AF by increasing the activity of the inward rectifier K+ channel. 2005 Elsevier Inc. All rights reserved. Keywords: Kir2.1; KCNJ2; Ion channel; Atrial fibrillation; Molecular genetics
As the most common cardiac arrhythmia in clinical practice, atrial fibrillation (AF) is characterized by rapid and irregular activation of the atrium. AF prevalence increases progressively with age, with nearly 6% in those over 65 years of age. A recent Framingham Heart Study showed that lifetime risks for AF development were about 25% for men and women 40 years of age and older. AF may cause strokes and heart failures, and is a source of considerable morbidity and mortality [1–4]. However, to date, a definitive treatment approach has *
1
Corresponding author. Fax: +86 21 66371663. E-mail address: [email protected] (Y. Chen). These authors contributed equally to this work.
0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.054
not been established. According to a large survey performed in the arrhythmia clinic at Mayo Clinic in Rochester, Minnesota, about 36% patients with AF had no evident pathogeny, and 5% had a positive family history [5]. This points to the fact that a subset of AF has an important genetic background. Recently, we found two causative genes (K+ channel genes KCNQ1 and KCNE2) for familial AF [6,7]. However, more molecular basis or causative genes remain to be identified [5–10]. Understanding the electrophysiological process leading to AF has been a subject of intensive study over last century. Dobrev et al. [11,12] observed that the current density of IK1 in human atrial myocytes was twofold
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
greater during AF than in sinus rhythm. Van Wagoner et al. [13,14] and Bosch et al. [15] also noted a significant increase in IK1 in atrial myocytes of patients with AF. Kir2.1 to Kir2.4 subfamily of inward rectifier K+ channel family underlies cardiac IK1 [16,17]. Kir2.1 is encoded by KCNJ2, a causative gene of Andersen syndrome, which is characterized by a triad of periodic paralysis, cardiac arrhythmia, and skeletal developmental abnormalities [18]. The role of Kir2.1 in forming cardiac IK1 was supported by the absence of IK1 current in cardiac myocytes from Kir2.1/ mice [19]. The overexpression of Kir2.1 in the mouse heart upregulated IK1 and ultimately initiated multiple abnormalities of cardiac excitability including AF [20]. In this study, we analyzed the Kir2.1 coding gene KCNJ2, the congenital long QT syndrome-associated genes (KCNQ1, HERG, SCN5A ANK-B, KCNE1, and KCNE2), and several other channel genes (KCNE3, KCNE4, and KCNE5) in 30 unrelated Chinese kindreds with AF. We found a missense mutation in KCNJ2 in one Chinese family. Heterologous expression in COS and HEK cells revealed a gain-of-function effect consistent with the AF clinical manifestations seen in this family.
Materials and methods Subjects. We identified 30 unrelated kindreds of familial AF among Chinese Han population. The diagnostic criteria for familial AF were a positive AF family history, electrocardiographic evidence of AF, and exclusion of organic heart diseases and other causative factors. We screened 10 ion channel or transporter related genes (KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5) (GenBank Accession Nos. AJ006345, NT_007914, NT_022517, NW_105991, AP001720, NM_005136, NT_035430, XM_208561, NT_005403, and NM_012282. GenBank: http:// www.ncbi.nlm.nih.gov/GenBank) in these 30 probands and in their family members. The controls were 420 unrelated healthy Chinese subjects of Han nationality. The study was approved by the Ethical Committee of Chinese Human Genome Center at Shanghai. Written informed consent was obtained from all subjects. Genetic analysis. DNA was extracted from whole venous blood with Wizard Genomic DNA Purification Kit (Promega). All coding sequences of the KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5 were amplified using HotStarTaq DNA Polymerase (Qiagen). Products of PCR amplification were purified with MBI Fermentas DNA Extraction Kit (MBI). Both strands of each PCR product were sequenced with a DYEnamic ET dye terminator kit (Amersham Biosciences) under MegaBACE 500 DNA Sequencing system (Amersham Biosciences). RT-PCR. For RT-PCR, human atrial and ventricular tissue specimens were collected and preserved in RNAlater RNA stabilization reagent (Qiagen). Total RNA was prepared using an RNeasy Protect Mini kit (Qiagen). An on-column DNase treatment was included in the RNA isolation step. Before RT-PCR, RNA samples were further treated with DNase (Invitrogen). RT was performed with KCNJ2 (5 0 GTGACACATCTGAAACCATAGCC-3 0 ) and control gene GAPDH (5 0 -CCACCACCCTGTTGCTGTAG-3 0 ) specific primers using SuperScriptIIreverse transcriptase (Invitrogen). PCR was performed in a 25 ll reaction mixture with an annealing temperature at 62 C, and
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35 cycles. Q-solution was included in the reactions for the KCNJ2 and GAPDH transcripts. The primer pairs used for the concurrent amplification of the two transcripts were for KCNJ2: forward—5 0 TCAGTAGACAGACCTTGGTAGAACC-3 0 , reverse—5 0 -GTGA CACATCTGAAACCATAGCC-3 0 , product size 621 bp; for GAPDH: forward—5 0 -GCCAAGGTCATCCATGACA-3 0 , reverse—5 0 -CCAC CACCCTGTTGCTGTAG-3 0 , product size 496 bp. Genomic DNAnegative control for PCR was conducted by adding RNA template instead of RT product to the PCR mixture. Mutagenesis. Forward (5 0 -GCAGGATCCAAAGCAGAAGCA CTGGAGTC-3 0 ) and reverse (5 0 -GCAGCGGCCGCTGACCCAT CTTGACCAGTACC-3 0 ) primers were used to amplify the coding regions and untranslated flanking regions of the human KCNJ2 gene using pfuUltra high-fidelity DNA polymerase (Stratagene). Purified, BamHI and NotI digested PCR product was subcloned into the BamHI and NotI sites of the pXOOM expression vector (kindly provided by Dr. T Jespersen, University of Copenhagen, Denmark). Site-directed mutagenesis was performed using the QuickChange II XL Site-Directed Mutageneses Kit. The forward and reverse primers were 5 0 -ATCTTCTGCCTGGCTTTCATCCTGTCATGGCTGTT TT-3 0 and 5 0 -AAAACAGCCATGACAGGATGAAAGCCAGGCA GAAGAT-3 0 , respectively. The entire coding region of the mutant clone was confirmed by sequencing. Transfection and electrophysiology. Cells (COS-7 or HEK293) were maintained as described [6]. Cells were transiently transfected by DEAE–dextran precipitate method with 2 lg wild-type (WT) KCNJ2pXOOM DNA or 2 lg mutant-type (MT) KCNJ2-pXOOM DNA or 1 lg WT and 1 lg MT KCNJ2-pXOOM DNA per 60-mm culture dish. Current recordings were performed 48 h after transfection in the whole-cell configuration at room temperature (22 C), using EPC-10 amplifier (HEKA Instruments), and as previously described [6]. Confocal microscopy. In order to monitor Kir2.1 subcellular localization, WT KCNJ2 and MT KCNJ2 were transferred into the pEYFP-N1 vector (enhanced yellow fluorescent protein, Clontech) and the pECFP-N1 vector (enhanced cyan fluorescent protein, Clontech), respectively. The whole coding region of the two plasmid constructs was confirmed by sequencing. Fluorescence microscopy was performed with a Leica confocal microscopy system. Statistical analysis. Current changes were assessed by means of ANOVA and DunnettÕs t test. All reported p values are two-sided and a p value of 0.05 or less was considered to indicate statistical significance. Data are given as means ± SE.
Results Identification of Kir2.1 V93I mutation The probands in 30 kindreds of familial AF were clinically and genetically evaluated. None of them had evident etiologies for AF. We sequenced 10 ion channel or transporter related genes (KCNQ1, HERG, SCN5A, ANK-B, KCNJ2, KCNE1, KCNE2, KCNE3, KCNE4, and KCNE5) after PCR amplification of genomic DNA. A novel missense mutation in KCNJ2 was found in one of the probands. With several DNA samples unavailable and the phenotypes of II:12, III:3, and III:6 uncertain, using the flanking microsatellite D17S949, a two-point linkage analysis between KCNJ2 and the familial AF gave a LOD score of 1.93 at recombination fraction 0 (detailed data not shown). The proband (II-3) who had paroxysmal AF
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as frequently as two to three times a month was a heterozygote for the 277G fi A transition, corresponding to a V93I substitution in KCNJ2. His father (I-1) and older sister (II-2) had permanent AF before death. V93I was also found in his younger sister (II-6), niece (III-2), and nephews (III-3 and III6). His younger sister (II-6) experienced paroxysmal AF as frequently as once or twice a week. His niece (III-2) showed paroxysmal AF on 24-h electrocardiographic recordings. His nephews (III-3 and III-6 who was 42 and 33 years old, respectively) did not have AF on 24-h electrocardiographic monitoring. In view of the characteristic of paroxysmal onset of AF, we did not exclude that III3 and III-6 were AF patients. We did not obtain the blood sample of II-12 and did not know her phenotype. Other unaffected family members did not carry the V93I substitution. The substitution co-segregated with AF in the family with the exception of III-3 and III-6 (Table 1 and Fig. 1). All affected members in this family had normal QT interval on 12-lead ECG, experienced no episodes of muscle weakness or syncope, and did not have frequent premature ventricular contractions or ventricular tachycardia on 24-h electrocardiographic monitoring. Serum potassium levels were within normal limits. None of the patients exhibited any developmental problems such as cleft palate, low set ears, short stature, clinodactyly, syndactyly, or brachydactyly, as reported for AndersenÕs syndrome. To further confirm that the V93I substitution of Kir2.1 was not a benign polymorphism, we checked this substitution in 420 healthy individuals. It was not found in any of these healthy individuals. Kir2.1 expression in human atrium Although Kir2.1 is expressed in human heart, its distribution in different heart chambers is not clear. Qualitative RT-PCR was performed to determine the tissue distribution of KCNJ2 mRNA in the human heart. Our data showed that Kir2.1 was strongly expressed in both ventricles and atria, with lower signals in atria (Fig. 1).
Conservation of sequences flanking Kir2.1 V93I mutation Kir2.1 subunits are made of two membrane-spanning segments (M1 and M2) adjacent to a pore-forming loop, and cytoplasmic N- and C-termini. V93I substitution is located in the outer helix of the M1 domain [21] which displays a highly conserved sequence among human, domestic guinea pig, pig, dog, cow, Norway rat, rabbit, and house mouse (Table 2 and Fig. 2). V93I substitution perturbs a highly conserved residue suggesting that V93I may have important functional consequences. Functional effect of V93I mutation on Kir2.1 channel We expressed WT and MT KCNJ2 in COS-7 cells, and performed electrophysiological study using wholecell patch-clamping technique. The data were as follows: (1) the currents for MT expression at potentials ranging from 140 to 80 mV or from 60 to 40 mV had amplitudes significantly higher than those for WT expression. For example, at 90 mV, the current for MT expression was 97 ± 7.8 pA/pF (n = 20) and that for WT expression 40 ± 4.9 pA/pF (n = 22) (p < 0.01); whereas at 50 mV, the former was 97 ± 8.0 pA/pF (n = 20) and the latter 15 ± 3.0 pA/pF (n = 22) (p < 0.001) (Fig. 3); (2) at the same potential range, co-expression of MT and WT also significantly enhanced the Kir2.1 current (Fig. 3); (3) compared with the currents for co-expression of MT and WT, the currents for MT expression at potentials ranging from 50 to 40 mV were significantly larger (Fig. 3). Similar results were obtained in HEK293 cells (data not shown). Hence, it is concluded that the V93I mutation had a gain-of-function effect on Kir2.1 channels. Subcellular trafficking of mutant Kir2.1 channel To assess the subcellular trafficking of WT and MT KCNJ2 subunits, we performed confocal laser scanning microscopy in COS-7 and HEK293 cells. Cells transfected with WT KCNJ2 showed the same plasma membrane
Table 1 Clinical characteristics of subjects with Kir2.1 V93I mutation Family member
Sex
Age (year)
Age at AF diagnosis (year)
Recurrent palpitation
AF classification
Ventricular rate (bpm)
QTc (s)
LAD (mm)
LVEF (%)
I-1 II-2 II-3 II-6 III-2 III-3 III-6
M F M F F M M
82a 56a 59b 56b 57b 42b 33b
58 50 54 50 57 NA NA
+ + + +
Permanent Permanent Paroxysmal Paroxysmal Paroxysmal NA NA
NA 63/70c 68/110c 71/118c 66/102c 83 66
NA 0.42/0.43c 0.41/0.44c 0.43/0.43c 0.42/0.43c 0.41 0.37
NA 35 36 35 31 33 34
NA 73 65 66 71 68 65
QTc, corrected QT interval; NA, not available or not applicable; LAD, left atrial dimension; LVEF, left ventricular ejection fraction. a The age at death is shown. b Age is the current age. c The value in AF is shown.
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
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Fig. 1. Familial AF associated with Kir2.1 V93I mutation. (A) Pedigree for a Chinese AF kindred. Asterisks indicate that the individual had the V93I mutation. (B) Normal KCNJ2 DNA sequence. (C) KCNJ2 DNA sequence of the affected family members. Sequence analysis of DNA from the affected family members after PCR amplification revealed a G-to-A substitution at nucleotide 277 in KCNJ2 causing a V93I mutation. (D) RT-PCR results for transcripts of KCNJ2 and GAPDH genes concurrently expressed in human atrial and ventricular tissues.
fluorescence pattern as those transfected with MT KCNJ2. In cells co-transfected with WT and MT KCNJ2, both proteins also demonstrated a similar fluorescence
pattern (data not shown). Tagging with the fluorescent protein did not affect the Kir2.1 channel function (data not shown).
Table 2 Conservation of sequences flanking Kir2.1 V93I mutation Species
Amino acids flanking residue 93
Identitiesa (%)
Human Mutant V931 Domestic guinea pig Pig Dog Cow Norway rat Rabbit House mouse
KGQRYLADIFTTCVDIRWRWMLVIFCLAFVLSWLFFGCVFWLIALLHGDLDASK ...............................................................................I............................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... ............................................................................................................................................... .............................................................................................................................................R ..........................................................................................................................................T...
99 99 99 99 99 99 98
a
Identity is the percentage of the sequence that is identical to human sequence as based on protein sequences from the following GenBank ID (gi): Human Kir2.1, gi: 4504835; domestic guinea pig Kir2.1, gi: 1708549; pig Kir2.1, gi: 3024031; dog Kir2.1, gi: 13878561; Cow Kir2.1, gi: 27805969; Norway rat Kir2.1, gi: 2493597; rabbit Kir2.1, gi: 1352481; house mouse Kir2.1, gi: 547735.
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Fig. 2. The proximity of residue valine 93 to the Kir2.1 channel pore. Residue V93 on the Kir2.1 sequence aligns to residue V72 on the KirBac1.1 crystal structure and is located in the outer helix.
Discussion The availability of a large number of characterized AF kindreds in our cardiology center allowed us to identify KCNJ2 as a novel gene for familial AF. Interestingly, this gene has already been linked to AndersenÕs syndrome [22]. Unlike loss-of-function mutations in AndersenÕs syndrome, the V93I substitution identified in our AF family had a Kir2.1 gain-of-function effect. This mutation was present in all affected family members and absent in 420 healthy subjects. It was reported that overexpression of wild-type Kir2.1 in the mouse heart led to AF and other abnormalities of cardiac excitability [20]. Thus, we hypothesized that Kir2.1 mutations might be associated with familial AF. Before the Kir2.1 coding gene KCNJ2 was sequenced, we performed two-point linkage analysis on the four-generation family. Although several DNA samples were unavailable and the phenotypes of II:12, III:3, and III:6 uncertain, a LOD score of 1.93 was obtained at recombination fraction zero at D17S949 (data not shown). To date, over 20 Kir2.1 mutations have been found in families with AndersenÕs syndrome, all of which resulted in loss-of-function with dominant-negative suppression of inward currents, except for two, which displayed haplo-insufficiency [22–26]. In contrast, V93I had a gain-of-
function effect on Kir2.1 channels. The functional expression of the AF-associated V93I substitution demonstrated a significant gain-of-function in both inward and outward Kir2.1 currents, without affecting the kinetics and rectification properties. Although not quantified, the results with the fluorescently labeled Kir2.1 subunits are in favor of a normal trafficking of the mutated channels. A direct effect on channel conductance is more likely, as suggested by the proximity of residue valine 93 to the pore (Fig. 2). Kir2.1 plays a major role in both the late phase of repolarization (phase 3) and the phase of resting membrane potential (phase 4), but almost no current is allowed to pass the channel during the plateau phase of action potential [16,27–31]. At potentials negative to resting membrane potential, inward rectifier channels pass a large inward current to bring the potential back to the resting status. In contrast, at potentials positive to the resting membrane potential, inward rectifier channels pass a much smaller outward current to repolarize the cell. The activated inward rectifier channels may amplify and accelerate the rate of return of the membrane to its resting membrane potential value, leading to shortening of action potential duration [30,32,33]. As expected, the Kir2.1 loss-of-function mutations led to QT interval prolongation in AndersenÕs syndrome patients. Kir2.1 V93I mutation increased inward potassium current at 90 to 80 mV and outward potassium current at 60 to 40 mV. These would stabilize resting membrane potential and shorten the repolarization phase of the atrial action potential, resulting in a shortening of the atrial effective refractory period (ERP). Thus, the gain-of-function effect of V93I mutation may create a substrate favorable to a multiple wavelet re-entry, a dominant mechanism of AF [2]. An overexpression of Kir2.1 in mouse heart upregulated IK1 and initiated AF [20]. IK1 increase has also been reported in human acquired AF [11–14]. Additionally, all the two human AF-associated mutations reported to date increased K+ channel current [6,7]. The gain-of-function mutation of cardiac K+ channels could be a good trigger for AF. New evidence has been found to support the speculation. A gain-of-function mutation of HERG, a K+ channel, caused not only short QT syndrome but also AF [34]. According to the protein sequence alignment, the sequences flanking the KCNJ2 V93I substitution are well conserved in all mammals with KCNJ2 sequence known. However, the substitution can be found in birds (chickens and domestic pigeons) (GenBank Accession Nos.: chicken KCNJ2, gi: 45382445; domestic pigeon KCNJ2, gi: 6273353). This does not seem to support our hypothesis that KCNJ2 V93I is a novel mutation associated with familial AF. In view of this, we analyzed the expression of KCNJ2 in the hearts of chicken and domestic pigeon. We found that KCNJ2 was not
M. Xia et al. / Biochemical and Biophysical Research Communications 332 (2005) 1012–1019
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Fig. 3. Functional expression of Kir2.1 V93I mutation in COS-7 cells. (A) Representative Kir2.1 currents. WT, currents from COS-7 cells transfected with wild Kir2.1 channels; WT + MT, currents from COS-7 cells co-transfected with wild and mutant Kir2.1 channels; MT, currents from COS-7 cells transfected with mutant Kir2.1 channels. (B) Kir2.1 current–voltage relationship. It was obtained from COS-7 cells expressing WT–WT Kir2.1 protein (j) (n = 22), WT–MT Kir2.1 protein (m) (n = 18), and MT–MT Kir2.1 protein (d) (n = 20). Currents were elicited by holding the cells to 80 mV, then cells were subjected to test pulses ranging from 140 to +50 mV for 200 ms. Values are means ± SE.
expressed in the atria of these two birds (data not shown). Thus, KCNJ2 may not participate in the physiological activity of the atrium in birds such as chicken and domestic pigeon, although it does in mammals. The appearance of the substitution in chicken and domestic pigeon does not conflict with the association of V93I with familial AF. Not all mutation carriers exhibited AF. The carriers (III-3 and III-6) did not have AF on a 24-h electrocardiographic monitoring, which may be explained by any or a combination of the following reasons: (1) these carriers were relatively young (33 and 42 years old) while AF usually occurs in aged patients; (2) paroxysmal AF occurs as rarely as a few times in life in some patients [3]. A longer duration of electrocardiographic monitoring may be required to record paroxysmal AF in these patients; (3) the familial AF caused by V93I mutation may have a low penetrance; (4) V93I may be only a genetic predisposing factor for AF. Environmental factors may also be involved in the onset of AF. We analyzed 154 patients with lone AF (data not shown). V93I mutation was not found in this popula-
tion, indicating that more linked genes remain to be identified in most of the AF patients without structural heart diseases. Kir2.1 gain-of-function mutation is one of the AF molecular bases. The study not only identifies a molecular mechanism of the genetic form of AF, but also sheds light on molecular mechanism of the acquired forms of AF. Kir2.1 channels or IK1 currents may be potential targets for drug therapy of AF.
Acknowledgments We are indebted to the family with AF for participating in the study. This work was supported by grants from the National Science Fund for Distinguished Young Scholars of China (30425016), the National Natural Science Foundation of China (30330290, 30170386, and 39900060), the Major Program Fund by the Ministry of Education of China (104081), the Shanghai Science and Technology Development Fund of China (SKW0202), the Shanghai Dawn Scholar Fund of China
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(SKW0103), and the Centre National de la Recherche Scientifique Fund of France.
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