Functional Characterization of a Trafficking-defective HCN4 Mutation, D553N, Associated with Cardiac Arrhythmia

Abstract

Hyperpolarization-activated cyclic nucleotide-gated channel 4 gene HCN4 is a pacemaker channel that plays a key role in automaticity of sinus node in the heart, and an HCN4 mutation was reported in a patient with sinus node dysfunction. Expression of HCN4 in the heart is, however, not confined to the sinus node cells but is found in other tissues, including cells of the conduction system. On the other hand, mutations in another cardiac ion channel gene, SCN5A, also cause sinus node dysfunction as well as other cardiac arrhythmias, including long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, and progressive cardiac conduction disturbance. These observations imply that HCN4 abnormalities may be involved in the pathogenesis of various arrhythmias, similar to the SCN5A mutations. In this study, we analyzed patients suffering from sinus node dysfunction, progressive cardiac conduction disease, and idiopathic ventricular fibrillation for mutations in HCN4. A missense mutation, D553N, was found in a patient with sinus node dysfunction who showed recurrent syncope, QT prolongation in electrocardiogram, and polymorphic ventricular tachycardia, torsade de pointes. In vitro functional study of the D553N mutation showed a reduced membranous expression associated with decreased If currents because of a trafficking defect of the HCN4 channel in a dominant-negative manner. These data suggest that the loss of function of HCN4 is associated with sinus nodal dysfunction and that a consequence of pacemaker channel abnormality might underlie clinical features of QT prolongation and polymorphic ventricular tachycardia developed under certain conditions. Hyperpolarization-activated cyclic nucleotide-gated channel 4 gene HCN4 is a pacemaker channel that plays a key role in automaticity of sinus node in the heart, and an HCN4 mutation was reported in a patient with sinus node dysfunction. Expression of HCN4 in the heart is, however, not confined to the sinus node cells but is found in other tissues, including cells of the conduction system. On the other hand, mutations in another cardiac ion channel gene, SCN5A, also cause sinus node dysfunction as well as other cardiac arrhythmias, including long QT syndrome, Brugada syndrome, idiopathic ventricular fibrillation, and progressive cardiac conduction disturbance. These observations imply that HCN4 abnormalities may be involved in the pathogenesis of various arrhythmias, similar to the SCN5A mutations. In this study, we analyzed patients suffering from sinus node dysfunction, progressive cardiac conduction disease, and idiopathic ventricular fibrillation for mutations in HCN4. A missense mutation, D553N, was found in a patient with sinus node dysfunction who showed recurrent syncope, QT prolongation in electrocardiogram, and polymorphic ventricular tachycardia, torsade de pointes. In vitro functional study of the D553N mutation showed a reduced membranous expression associated with decreased If currents because of a trafficking defect of the HCN4 channel in a dominant-negative manner. These data suggest that the loss of function of HCN4 is associated with sinus nodal dysfunction and that a consequence of pacemaker channel abnormality might underlie clinical features of QT prolongation and polymorphic ventricular tachycardia developed under certain conditions. Heart rate is regulated by automaticity of sinus node cells. Sinus node dysfunction (SND) 1The abbreviations and trivial terms used are: SND, sinus node dysfunction; IVF, idiopathic ventricular fibrillation; LQTS, long QT syndrome; HCN, hyperpolarization-activated cyclic nucleotide-gated; CNBD, cyclic nucleotide binding domain; RT-PCR, reverse transcription and polymerase chain reaction; GFP, green fluorescent protein; V½, half maximal voltage; rab-WT, wild type rabbit; mut-rab, mutant rabbit; RT, reverse transcription; If, pacemaker funny current. is a type of cardiac arrhythmia seen relatively infrequently, and a wide variety of clinical symptoms are noticed, from mild to severe, including fatigue, palpitations, anxiety, dizziness, fainting, and syncope in some cases. Physical signs of SND can also be found as inadequate heart rate at rest or in response to exercise. Although dysfunction of the sinus node automaticity is often associated with acquired cardiac conditions such as ischemic heart disease, cardiomyopathy, congestive heart failure, or metabolic diseases, there are a few patients with idiopathic SND who do not suffer from these disease conditions. In patients with idiopathic SND, genetic abnormalities may be found in ion channel genes, similar to the cases with other inherited cardiac arrhythmias such as long QT syndrome (LQTS), Brugada syndrome and idiopathic ventricular fibrillation (IVF), and progressive cardiac conduction disturbance (1Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (1996) Google Scholar, 2Wang Q. Shen J. Splawski I. Atkinson D. Li Z. Robinson J.L. Moss A.J. Towbin J.A. Keating M.T. Cell. 1995; 80: 805-811Abstract Full Text PDF PubMed Scopus (1436) Google Scholar, 3Wang Q. Curran M.E. Splawski I. Burn T.C. Millholland J.M. Van-Raay T.J. Shen J. Timothy K.W. Vincent G.M. de Jager T. Schwartz P.J. Towbin J.A. Moss A.J. Atkinson D.L. Landes G.M. Connors T.D. Keating M.T. Nat. Genet. 1996; 12: 17-23Crossref PubMed Scopus (1501) Google Scholar, 4Splawski I. Tristani-Firouzi M. Lehmann M.H. Sanguinetti M.C. Keating M.T. Nat. Genet. 1996; 17: 338-340Crossref Scopus (676) Google Scholar, 5Chen Q. Kirsch G.E. Zhang D. Brugada R. Brugada J. Brugada P. Potenza D. Moya A. Borggrefe M. Breithardt G. Ortiz-Lopez R. Wang Z. Antzelevitch C. O'Brien R.E. Schulze-Bahr E. Keating M.T. Towbin J.A. Wang Q. Nature. 1998; 392: 293-296Crossref PubMed Scopus (1540) Google Scholar, 6Abbott G.W. Sesti F. Splawski I. Buck M.E. Lehmann M.H. Timothy K.W. Keating M.T. Goldstein S.A. Cell. 1999; 97: 175-187Abstract Full Text Full Text PDF PubMed Scopus (1177) Google Scholar, 7Mohler P.J. Schott J.J. Gramolini A.O. Dilly K.W. Guatimosim S. duBell W.H. Song L.S. Haurogne K. Kyndt F. Ali M.E. Rogers T.B. Lederer W.J. Escande D. Le Marec H. Bennett V. Nature. 2003; 421: 634-639Crossref PubMed Scopus (835) Google Scholar, 8Plaster N.M. Tawil R. Tristani-Firouzi M. Canun S. Bendahhou S. Tsunoda A. Donaldson M.R. Iannaccone S.T. Brunt E. Barohn R. Clark J. Deymeer F. George Jr., A.L. Fish F.A. Hahn A. Nitu A. Ozdemir C. Serdaroglu P. Subramony S.H. Wolfe G. Fu Y.H. Ptacek L.J. Cell. 2001; 105: 511-519Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 9Moss A.J. Schwartz P.J. Crampton R.S. Tzivoni D. Locati E.H. MacCluer J. Hall W.J. Weitkamp L. Vincent G.M. Garson Jr., A. Circulation. 1991; 84: 1136-1144Crossref PubMed Scopus (845) Google Scholar, 10Akai J. Makita N. Sakurada H. Shirai N. Ueda K. Kitabatake A. Nakazawa K. Kimura A. Hiraoka M. FEBS Lett. 2000; 479: 29-34Crossref PubMed Scopus (117) Google Scholar, 11Priori S.G. Napolitano C. Tiso N. Memmi M. Vignati G. Bloise R. Sorrentino V.V. Danieli G.A. Circulation. 2001; 103: 196-200Crossref PubMed Scopus (1152) Google Scholar, 12Marban E. Nature. 2002; 415: 213-218Crossref PubMed Scopus (326) Google Scholar, 13Splawski I. Shen J. Timothy K.W. Lehmann M.H. Priori S. Robinson J.L. Moss A.J. Schwartz P.J. Towbin J.A. Vincent G.M. Keating M.T. Circulation. 2000; 102: 1178-1185Crossref PubMed Scopus (1086) Google Scholar). One disease gene for SND is SCN5A, and its homozygous loss of function mutation leading to reduced cellular excitability predisposes to congenital sick sinus syndrome (14Benson D.W. Wang D.W. Dyment M. Knilans T.K. Fish F.A. Strieper M.J. Rhodes T.H. George Jr., A.L. J. Clin. Investig. 2003; 112: 1019-1028Crossref PubMed Scopus (459) Google Scholar). The other is the HCN4 mutation (15Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (313) Google Scholar), where a truncation mutation causes loss of exercise-induced increase of heart rate. SCN5A encodes an α-subunit of the cardiac sodium channel carrying the current to form a rapid upstroke of action potential (16Wang Q. Li Z. Shen J. Keating M.T. Genomics. 1996; 34: 9-16Crossref PubMed Scopus (271) Google Scholar), whereas HCN4 codes for an α-subunit of hyperpolarization-activated cation channel (17Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (314) Google Scholar). Because HCN4 is mainly expressed in sinus node cells and forms the pacemaker current (If), it is called the pacemaker channel (18Seifert R. Scholten A. Gauss R. Mincheva A. Lichter P. Kaupp U.B. Proc. Nat. Acad. Sci. U. S. A. 1999; 96: 9391-9396Crossref PubMed Scopus (229) Google Scholar). The HCN4 channel is a member of the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels activated by membrane hyperpolarization. HCN channels have evolutionary conserved structure in the core transmembrane domain, cyclic nucleotide binding domain (CNBD), and linker region connecting the core domain to the CNBD (19Wainger B.J. DeGennaro M. Santoro B. Siegelbaum S.A. Tibbs G.R. Nature. 2001; 411: 805-810Crossref PubMed Scopus (402) Google Scholar, 20Biel M. Schneider A. Wahl C. Trends Cardiovasc. Med. 2002; 12: 206-212Crossref PubMed Scopus (207) Google Scholar). The core domain consists of six transmembrane segments containing a pore-forming P region with a glycinetyrosine-glycine (GYG) sequence conserved in the selectivity filter among K+ channels. The HCN channels are quite diverse from the other K+ channels outside the GYG sequence, which may account for the properties of low K+ selectivity and voltage dependence for activation. According to these unusual channel properties, the HCN channels conduct a net inward current carried largely by Na+ at diastolic potential levels. Disturbance of the channel function in the heart may result in cardiac arrhythmias of life-threatening nature in some cases. Dysfunction of HCN channels in HCN2 or HCN4 knock-out mice causes sinus dysrhythmia because of the reduction of If current (21Stieber J. Herrmann S. Feil S. Loster J. Feil R. Biel M. Hofmann F. Ludwig A. Proc. Nat. Acad. Sci. U. S. A. 2003; 100: 15235-15240Crossref PubMed Scopus (376) Google Scholar, 22Ludwig A. Budde T. Stieber J. Moosmang S. Wahl C. Holthoff K. Langebartels A. Wotjak C. Munsch T. Zong X. Feil S. Feil R. Lancel M. Chien K.R. Konnerth A. Pape H.C. Biel M. Hofmann F. EMBO J. 2003; 22: 216-224Crossref PubMed Scopus (424) Google Scholar). In addition, expression of HCN4 is not restricted to sinus node cells in mice (23Moosmang S. Stieber J. Zong X. Biel M. Hofmann F. Ludwig A. Eur. J. Biochem. 2001; 268: 1646-1652Crossref PubMed Scopus (354) Google Scholar), implying that there would be some other phenotypes associated with HCN4 mutations. Here we have characterized a novel HCN4 mutation found in a patient with SND, QT prolongation, and polymorphic ventricular tachycardia, torsade de pointes and explored the mechanism of channel dysfunction caused by the mutation. This is the first report of a trafficking-defective HCN4 channel mutation. Expression of HCN4 in the Human Heart—Expression of HCN4 in the human heart was investigated semiquantitatively by reverse transcription and polymerase chain reaction (RT-PCR) using a human cardiovascular multiple tissue cDNA panel (Clontech). Each tissue cDNA, normalized to the expression levels of several different housekeeping genes by the manufacturer, was used as template for PCR amplification. RT-PCR was done with a pair of HCN4-specific primers of 5′-CCCGCCTCATTCGATATATTCAC-3′ and 5′-GAGCGCGTAGGAGTACTGCTTC-3′ (17Ludwig A. Zong X. Stieber J. Hullin R. Hofmann F. Biel M. EMBO J. 1999; 18: 2323-2329Crossref PubMed Scopus (314) Google Scholar), and the products were subjected to electrophoresis in a 1.5% agarose gel. The density of each PCR fragment was measured after staining with ethidium bromide (ATTO Corp.). Subjects—We analyzed 6, 3, and 16 genetically unrelated index patients with SND, progressive cardiac conduction disturbance, and IVF, respectively. Blood samples were obtained from each patient after an informed consent for gene analysis was received. These patients were first analyzed for mutations in the channel genes, including KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and parts of RYR2 (exons 41–49 and 76–105), by single-strand conformational polymorphism (SSCP) analysis (24Nishi H. Kimura A. Harada H. Toshima H. Sasazuki T. Biochem. Biophys. Res. Commun. 1992; 188: 379-387Crossref PubMed Scopus (43) Google Scholar) of PCR products obtained by using primers reported in the literature (8Plaster N.M. Tawil R. Tristani-Firouzi M. Canun S. Bendahhou S. Tsunoda A. Donaldson M.R. Iannaccone S.T. Brunt E. Barohn R. Clark J. Deymeer F. George Jr., A.L. Fish F.A. Hahn A. Nitu A. Ozdemir C. Serdaroglu P. Subramony S.H. Wolfe G. Fu Y.H. Ptacek L.J. Cell. 2001; 105: 511-519Abstract Full Text Full Text PDF PubMed Scopus (837) Google Scholar, 16Wang Q. Li Z. Shen J. Keating M.T. Genomics. 1996; 34: 9-16Crossref PubMed Scopus (271) Google Scholar, 25Splawski I. Shen J. Timothy K.W. Vincent G.M. Lehmann M.H. Keating M.T. Genomics. 1998; 51: 86-97Crossref PubMed Scopus (220) Google Scholar). No patients or family relatives showed clinical signs of Andersen syndrome, such as periodic paralysis and dysmorphic features, caused by KCNJ2 mutation (9Moss A.J. Schwartz P.J. Crampton R.S. Tzivoni D. Locati E.H. MacCluer J. Hall W.J. Weitkamp L. Vincent G.M. Garson Jr., A. Circulation. 1991; 84: 1136-1144Crossref PubMed Scopus (845) Google Scholar). The research protocol was approved by the Ethics Reviewing Committee of the Medical Research Institute, Tokyo Medical and Dental University. Mutational Analysis of HCN4—We designed primers to separately amplify eight coding exons of HCN4 (Fig. 2A). Each exon was amplified by using various combinations of primers. Sequences of the primers and conditions of PCR are available upon request. The PCR products from patients were searched for sequence variations by the single-strand conformational polymorphism method and subsequent direct sequencing. Distribution of GFP-tagged HCN4 Channel—Because human HCN4 cDNA was not available to us, we investigated functional changes caused by the D553N mutation using rabbit HCN4 cDNA (26Ishii T.M. Takano M. Xie L.H. Noma A. Ohmori H. J. Biol. Chem. 1999; 274: 12835-12839Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar). A mutation equivalent to the human HCN4 D553N was introduced into the wild type rabbit cDNA (rab-WT) to obtain a rabbit HCN4 D554N (rab-Mut). GFP-tagged HCN4 constructs were made from these constructs by replacing the termination codon with a GATATC sequence and the GFP sequence amplified with primers 5′-GATATCATGGCCAGCAAAGGAGAAGA-3′ and 5′-TCTAGACGCTTTTGTAGAGCTCATCCA-3′ from pcDNA3.1/NT-GFP (Invitrogen). These constructs were sequenced to ensure that no other mutation was introduced. The HCN4 constructs were transfected into COS7 cells. The amount of transfected DNA was 0.2 μg/well for each HCN4 construct (rab-WT: rab-Mut = 1:1 in the case of co-expression of rab-WT and rab-Mut) in a Lab-Tek 4 chamber mounted glass slide (Nalge Nunc International). The GFP signals from the transfected cells were examined using a confocal fluorescence microscope (Carl Zeiss AB). Electrophysiological Analysis of HCN4 Channel—COS7 cells were co-transfected with the construct of 0.4 μg of pEGFP-C1 (Clontech) and 1.6 μg of non-GFP-tagged rabbit HCN4 constructs. Cells were removed from culture dishes 36–48 h after the transfection and placed into a chamber on the stage of an inverted microscope (Diaphoto TMD; Nikon). The cells were superfused with Tyrode's solution containing 137 mm NaCl, 4 mm KCl, 1.8 mm CaCl2, 1 mm MgCl2, 10 mm glucose, and 10 mm HEPES (pH 7.4 adjusted with NaOH). Currents were recorded by a whole cell patch clamp configuration. Glass pipettes had an inner diameter of ∼1.0–1.5 μm and had resistance of 2–3 MΩ when filled with an internal solution composed of 110 mm K-gluconate, 20 mm KCl, 1 mm MgCl2, 5 mm EGTA, 5 mm MgATP, and 10 mm HEPES (pH 7.2 adjusted with KOH). After forming a whole cell configuration, cell membrane capacitance was estimated by analyzing capacity transient elicited by 5-mV hyperpolarizing pulses. Series resistance compensation was done by 50–70% using the circuit built into an amplifier (Axopatch 200B; Axon Instruments). A patch clamp amplifier was used to record membrane currents. Voltage-dependence of the If current activation was analyzed from tail currents by fitting to the Boltzmann equation of I/Imax = 1/(1 + exp[{V–V½}/κ]), where V½ and κ are half-maximal voltage for activation and slope factor, respectively. Software (pCLAMP8; Axon Instruments) was used to generate voltage protocols, to acquire data, and to analyze current signals. All experiments were done at 34 ± 1.0 °C. The data were obtained from 8–9 cells and represented by mean ± S.E. HCN4 Expression in the Human Heart—In a semiquantitative RT-PCR analysis, an HCN4-specific PCR product was detected not only in cDNA from an atrioventricular node but ubiquitously in cardiac tissue cDNAs (Fig. 1). Density of PCR product from each tissue was measured and compared with that from the atrioventricular node. Relative ratio was as follows: total adult heart, 0.24; total fetal heart, 0.35; aorta, 0.00; apex of the left ventricle, 0.78; left atrium, 0.90; right atrium, 1.10; right auricle, 1.33; left auricle, 0.67; left ventricle, 0.29; right ventricle, 0.35; and interventricular septum, 0.68. Mutational Analysis of HCN4 —To identify arrhythmia-related gene mutations in patients with cardiac arrhythmia, we first analyzed patients with SND, progressive cardiac conduction disturbance, and IVF for mutations in the channel genes KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2, and RYR2 by single-strand conformational polymorphism analysis and subsequent direct sequencing. Two SCN5A mutations were found in two IVF patients (details will be reported elsewhere), whereas no mutation was detected in the other patients; they were investigated further for the HCN4 mutation. The analysis of HCN4 revealed a novel mutation in a patient with SND that was not found in 380 control chromosomes, implying that it was not a polymorphism and might be a disease-associated mutation. The mutation was a G to A transition in exon 5, resulting in amino of with at codon D553N (Fig. A and This was in the linker region the core transmembrane domain and CNBD, which is an evolutionary conserved region among the HCN channel family (Fig. The index patient with D553N mutation was a who had from syncope for the first at of syncope at 34 upon a of the of showed of was and a cardiac for by polymorphic ventricular tachycardia, torsade de pointes (Fig. showed QT by heart rate was which was to by of or channel of D553N with the was found in family (Fig. and were for the mutation. Distribution of HCN4 with D553N a functional caused by the mutation, we compared of GFP-tagged channel of wild type rabbit HCN4 (rab-WT) and rabbit HCN4 D554N in transfected COS7 cells. D554N of rabbit HCN4 is equivalent to D553N of human HCN4 (Fig. expression of rab-WT was as fluorescence signals on the membrane (Fig. In only a of GFP-tagged rab-Mut was found on the cell and a amount of GFP was (Fig. of GFP-tagged rab-Mut with rab-WT showed GFP signals on the cell and (Fig. of GFP signals was reduced on the cell in the case of of GFP-tagged rab-WT with rab-Mut (Fig. These observations that the co-expression of mutant channels reduced the cell expression of channels in a dominant-negative manner. Electrophysiological Analysis of HCN4 with D553N of the mutant HCN4 channel were examined by a whole cell patch clamp method to COS7 cells transfected with GFP-tagged rabbit HCN4 constructs. hyperpolarizing were from a potential of and inward currents were activated at potential to in cells rab-WT, and tail currents were obtained at membrane potential to after various (Fig. The current for expressed rab-WT HCN4 was and the of current activation with of (Fig. and properties of inward currents recorded from transfected COS7 cells If current in sinus node cells D. PubMed Scopus Google Scholar). Because If and current we current in the and of to the HCN4 currents. the current was obtained in the of mm from the current in its (Fig. Cells transfected with rab-Mut showed decreased current with similar of activation as with of rab-WT and rab-Mut current half of rab-WT currents (Fig. The normalized tail current activation the (Fig. and its voltage dependence was analyzed by fitting to the Boltzmann equation to obtain various of channel in of V½ and slope factor, were not by the mutation, that voltage dependence in activation was not in the for current activation and were from in A and D. in activation was and in cells transfected with rab-Mut or in cells of COS7 cells transfected with various HCN4 constructs are as ± of of = = = ± ± ± ± ± ± in a ion channel dysfunction to gene mutations a of clinical of SCN5A mutations for SND, progressive cardiac conduction disturbance, Brugada syndrome, IVF, and This is not to the sodium ion channel for the gene, knock-out mice showed the SND J. R.S. Cell. Biol. 2003; PubMed Scopus Google in addition, mutations are well to cause (1Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (1996) Google Scholar, 10Akai J. Makita N. Sakurada H. Shirai N. Ueda K. Kitabatake A. Nakazawa K. Kimura A. Hiraoka M. FEBS Lett. 2000; 479: 29-34Crossref PubMed Scopus (117) Google Scholar, 12Marban E. Nature. 2002; 415: 213-218Crossref PubMed Scopus (326) Google Scholar). In this study, we an HCN4 mutation, D553N, in a patient suffering from recurrent cardiac syncope associated with QT and polymorphic ventricular Because no mutation in the genes for cardiac arrhythmia was found in the it was that the loss of function HCN4 mutation was associated with life-threatening cardiac arrhythmia in this HCN channels are expressed in cells of the sinus node and conduction such as the atrioventricular node and the it is not the expression of each HCN channel is confined to the cells of the conduction system. In this study, HCN4-specific RT-PCR product was found the cardiac tissues, that HCN4 is expressed in of various at the This is with a report on the cellular of HCN channels in the heart as by in (23Moosmang S. Stieber J. Zong X. Biel M. Hofmann F. Ludwig A. Eur. J. Biochem. 2001; 268: 1646-1652Crossref PubMed Scopus (354) Google Scholar). Although HCN4 expression in the ventricular is that in the atrioventricular the of the HCN4 channel in the to be as to its functional role in the cardiac conduction and The expressed current of rab-Mut and for HCN4 were decreased compared with that of rab-WT, whereas properties of current activation were not Although the activation of the mutant current were also the were decreased current function in this mutation. to decreased current in the confocal analysis of GFP-tagged channel that the mutation caused a trafficking a of and mutant HCN4 channels in to the cell trafficking of channels were reported for KCNH2, KCNE1, KCNQ1, SCN5A, and mutations to or Brugada syndrome J. Makita N. Sakurada H. Shirai N. Ueda K. Kitabatake A. Nakazawa K. Kimura A. Hiraoka M. FEBS Lett. 2000; 479: 29-34Crossref PubMed Scopus (117) Google Scholar, Z. Q. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, L. Shen Z. Priori S.G. Napolitano C. E. R. Schwartz P.J. Genet. 1999; PubMed Scopus Google Scholar). Because the trafficking defect of the mutant HCN4 channel was only in by HCN4 channel and expression of HCN4 channels was decreased by the co-expression of mutant HCN4 the HCN4 mutation as a dominant-negative HCN channels a role in diastolic 4 of cardiac action in cells and in sinus node cells D. PubMed Scopus Google Scholar, A. M. A. M. J. PubMed Scopus Google Scholar), heart rate by the or was reported that of an HCN4 channel in and QT prolongation in C. C. L. H. I. J. J.L. Eur. J. PubMed Scopus Google Scholar), the of on the and a A. M. D. J. 2002; PubMed Scopus Google Scholar). On the other hand, a mutation, of HCN4 was reported in a patient who showed a response of heart rate to prolongation of QT (15Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (313) Google Scholar). The mutation was to a channel the tail including CNBD, but the including structure would not be the mutant channel was to be expressed in transfected cells (15Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (313) Google Scholar). Because the mutant channel showed in its function for of response to the clinical of the patient was mild and the patient would not be in of life-threatening of cardiac syncope ventricular tachycardia was reported with the mutation (15Schulze-Bahr E. Neu A. Friederich P. Kaupp U.B. Breithardt G. Pongs O. Isbrandt D. J. Clin. Investig. 2003; 111: 1537-1545Crossref PubMed Scopus (313) Google Scholar). In the index patient with the D553N mutation showed and recurrent syncope, which that the trafficking abnormality of channels may the heart rate at is to be associated with QT and to to the of torsade de pointes J. B.J. R. Circulation. PubMed Scopus Google Scholar, Res. PubMed Scopus Google Scholar, T. T. N. N. H. S. M. K. J. 1992; Full Text PDF PubMed Scopus Google Scholar, Sanguinetti M.C. Res. PubMed Google Scholar), as was found in the patient with the D553N mutation, but the mechanism of such QT prolongation to be In we have for the first a trafficking-defective HCN4 mutation associated with life-threatening cardiac and functional in other patients are to the role of HCN4 mutations in various phenotypes of cardiac N. A. K. T. K. H. J. T. M. T. N. K. M. T. T. K. and J. for in samples and clinical from patients with or also M. Takano of for with rabbit HCN4