Keiko Tsuji

A Novel SCN5A Gain-of-Function Mutation M1875T Associated With Familial Atrial Fibrillation

OBJECTIVES This study describes a novel heterozygous gain-of-function mutation in the cardiac sodium (Na+) channel gene, SCN5A, identified in a Japanese family with lone atrial fibrillation (AF). BACKGROUND SCN5A mutations have been associated with a variety of inherited arrhythmias, but the gain-of-function type modulation in SCN5A is associated with only 1 phenotype, long-QT syndrome type 3 (LQTS3). METHODS We studied a Japanese family with autosomal dominant hereditary AF, multiple members of which showed an onset of AF or frequent premature atrial contractions at a young age. RESULTS The 31-year-old proband received radiofrequency catheter ablation, during which time numerous ectopic firings and increased excitability throughout the right atrium were documented. Mutational analysis identified a novel missense mutation, M1875T, in SCN5A. Further investigations revealed the familial aggregation of this mutation in all of the affected individuals. Functional assays of the M1875T Na(+) channels using a whole-cell patch-clamp demonstrated a distinct gain-of-function type modulation; a pronounced depolarized shift (+16.4 mV) in V(1/2) of the voltage dependence of steady-state inactivation; and no persistent Na+ current, which is a defining mechanism of LQTS3. These biophysical features of the mutant channels are potentially associated with increased atrial excitability and normal QT interval in all of the affected individuals. CONCLUSIONS We identified a novel SCN5A mutation associated with familial AF. The mutant channels displayed a gain-of-function type modulation of cardiac Na+ channels, which is a novel mechanism predisposing to increased atrial excitability and familial AF. This is a new phenotype resulting from the SCN5A gain-of-function mutations and is distinct from LQTS3.

Novel KCNJ2 Mutation in Familial Periodic Paralysis With Ventricular Dysrhythmia

Background—Mutations in the KCNJ2 gene, which codes cardiac and skeletal inward rectifying K+ channels (Kir2.1), produce Andersen’s syndrome, which is characterized by periodic paralysis, cardiac arrhythmia, and dysmorphic features. Methods and Results—In 3 Japanese family members with periodic paralysis, ventricular arrhythmias, and marked QT prolongation, polymerase chain reaction/single-strand conformation polymorphism/DNA sequencing identified a novel, heterozygous, missense mutation in KCNJ2, Thr192Ala (T192A), which was located in the putative cytoplasmic chain after the second transmembrane region M2. Using the Xenopus oocyte expression system, we found that the T192A mutant was nonfunctional in the homomeric condition. Coinjection with the wild-type gene reduced the current amplitude, showing a weak dominant-negative effect. Conclusions—T192, which is located in the phosphatidylinositol-4,5-bisphosphate binding site and also the region necessary for Kir2.1 multimerization, is a highly conserved amino acid residue among inward-rectifier channels. We suggest that the T192A mutation resulted in the observed electrical phenotype.

Cystic fibrosis transmembrane conductance regulator mediates sulphonylurea block of the inwardly rectifying K+ channel Kir6.1

1 Recombinant ATP‐sensitive K+ channels (KATP channels) were heterologously expressed in the NIH3T3 mouse cell line, and the electrophysiological properties were studied using patch‐clamp techniques. 2 The NIH3T3 cell lines transfected with the inwardly rectifying K+ channel Kir6.1 alone or with both Kir6.1 and cystic fibrosis transmembrane conductance regulator (CFTR) exhibited time‐independent K+ currents with weak inward rectification. In contrast, no measurable K+ conductance was observed in mock‐transfected cells or in cells transfected with CFTR alone. Regardless of co‐transfection with Kir6.1, the transfection with CFTR produced a Cl− conductance that was activated by cell dialysis with cAMP (1 mM). The conductance was reversibly suppressed by glibenclamide (30 μM). 3 Whole‐cell currents at +60 mV were blocked in a concentration‐dependent manner by Ba2+ ions with similar IC50 values: 89.3 ± 23.3 μM (Kir6.1 alone) and 67.3 ± 24.9 μM (Kir6.1‐CFTR). 4 The currents recorded from Kir6.1‐transfected cells were not affected by glibenclamide, whereas glibenclamide did inhibit the conductance expressed in cells co‐transfected with CFTR (IC50= 35.9 ± 6.6 μM). 5 In the cell‐attached mode with a 150 mM K+ pipette solution, both Kir6.1‐ and Kir6.1‐CFTR‐transfected cells displayed a class of K+ channels showing weak inward rectification and a slope conductance of 50.7 ± 1.0 and 52.4 ± 4.9 pS, respectively. 6 In the inside‐out mode, the single‐channel currents recorded from both types of cells were not inhibited by intracellular ATP (1 mM). However, glibenclamide was found to block the single‐channel activities in the co‐transfected cells.

Novel KCNE3 mutation reduces repolarizing potassium current and associated with long QT syndrome.

Long QT syndrome (LQTS) is an inherited disease involving mutations in the genes encoding a number of cardiac ion channels and a membrane adaptor protein. Among the genes that are responsible for LQTS, KCNE1 and KCNE2 are members of the KCNE family of genes, and function as ancillary subunits of Kv channels. The third KCNE gene, KCNE3, is expressed in cardiac myocytes and interacts with KCNQ1 to change the channel properties. However, KCNE3 has never been linked to LQTS. To investigate the association between KCNE3 and LQTS, we conducted a genetic screening of KCNE3 mutations and single nucleotide polymorphisms (SNPs) in 485 Japanese LQTS probands using DHPLC‐WAVE system and direct sequencing. Consequently, we identified two KCNE3 missense mutations, located in the N‐ and C‐terminal domains. The functional effects of these mutations were examined by heterologous expression systems using CHO cells stably expressing KCNQ1. One mutation, p.R99λH was identified in a 76‐year‐old woman who suffered torsades de pointes (TdP) after administration of disopyramide. Another mutation, p.T4A was identified in a 16‐year‐old boy and 67‐year‐old woman. Although the boy carried another KCNH2 mutation, he was asymptomatic. On the other hand, the woman suffered from hypokalemia‐induced TdP. In a series of electrophysiological analyses, the KCNQ1(Q1)+KCNE3(E3)‐R99λH channel significantly reduced outward current compared to Q1+E3‐WT, though the current density of the Q1+E3‐T4A channel displayed no statistical significance. This is the first report of KCNE3 mutations associated with LQTS. Screening for variants in the KCNE3 gene is of clinical importance for LQTS patients. Hum Mutat 30, 557–563, 2009.

Inhibitory actions of the phosphatidylinositol 3-kinase inhibitor LY294002 on the human Kv1.5 channel.

Background and purpose:  Kv1.5 channels conduct the ultra‐rapid delayed rectifier potassium current (IKur), and in humans, Kv1.5 channels are highly expressed in cardiac atria but are scarce in ventricles. Pharmacological blockade of human Kv1.5 (hKv1.5) has been regarded as effective for prevention and treatment of re‐entry‐based atrial tachyarrhythmias. Here we examined blockade of hKv1.5 channels by LY294002, a well‐known inhibitor of phosphatidylinositol 3‐kinase (PI3K).

Adrenergic regulation of the rapid component of delayed rectifier K+ current: Implications for arrhythmogenesis in LQT2 patients

BACKGROUND KCNH2 gene mutations disrupting rapid component of I(K) (I(Kr)) underlie type 2 congenital long QT syndrome (LQT2). Startled auditory stimuli are specific symptomatic triggers in LQT2, thus suggesting fast arrhythmogenic mechanism. OBJECTIVE We investigated acute alpha(1A)- and cyclic adenosine monophosphate (cAMP)-related beta-adrenergic modulation of I(Kr) in HL-1 cardiomyocytes, wild type (WT)- and 2 LQT2-associated mutant Kv11.1 channels (Y43D- and K595E-Kv11.1) reconstituted in Chinese hamster ovary (CHO) cells. METHODS I(Kr) and Kv11.1 currents were recorded using the whole-cell patch-clamp technique and confocal microscopy of HL-1 cardiomyocytes transfected with green fluorescent protein (GFP)-tagged pleckstrin homology domain of phospholipase C-delta(1) visualized fluctuations of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)) content. RESULTS In HL-1 cardiomyocytes expressing human alpha(1A)-adrenoceptor, superfusion with phenylephrine significantly reduced I(Kr) amplitude, shifted current activation to more positive potentials, and accelerated kinetics of deactivation. Confocal images showed a decline of membrane PIP(2) content during phenylephrine exposure. Simultaneous application of adenylyl cyclase activator forskolin and phosphodiesterase inhibitor 3-isobutyl-1-methylxantine (IBMX) shifted I(Kr) activation to more negative potentials and decreased tail current amplitudes after depolarizations between +10 and +50 mV. In CHO cells, alpha(1A)-adrenoceptor activation downregulated WT-Kv11.1 channels and forskolin/IBMX produced a dual effect. Expressed alone, the Y43D-Kv11.1 or K595E-Kv11.1 channel had no measurable function. However, co-expression of WT-Kv11.1 and each mutant protein evoked currents with loss-of-function alterations but identical to WT-Kv11.1 alpha(1A)- and forskolin/IBMX-induced regulation. CONCLUSION Acute adrenergic regulation of at least 2 Kv11.1 mutant channels is preserved as in WT-Kv11.1 and native I(Kr). Suppression of alpha(1A)-adrenoceptor-related transduction might have therapeutic implications in some cases of LQT2.

A Novel KCNJ2 Nonsense Mutation, S369X, Impedes Trafficking and Causes a Limited Form of Andersen-Tawil Syndrome

Background— Mutations in KCNJ2, a gene encoding the inward rectifier K+ channel Kir2.1, are associated with Andersen-Tawil syndrome (ATS), which is characterized by (1) ventricular tachyarrhythmias associated with QT (QU)-interval prolongation, (2) periodic paralysis, and (3) dysmorphic features. Methods and Results— We identified a novel KCNJ2 mutation, S369X, in a 13-year-old boy with prominent QU-interval prolongation and mild periodic paralysis. The mutation results in the truncation at the middle of the cytoplasmic C-terminal domain that eliminates the endoplasmic reticulum (ER)-to-Golgi export signal. Current recordings from Chinese hamster ovary cells transfected with KCNJ2-S369X exhibited significantly smaller K+ currents compared with KCNJ2 wild type (WT) (1 &mgr;g each) (−84±14 versus −542±46 picoamperes per picofarad [pA/pF]; −140 mV; P<0.0001). Coexpression of the WT and S369X subunits did not show a dominant-negative suppression effect but yielded larger currents than those of WT+S369X (−724±98 pA/pF>−[84+542] pA/pF; 1 &mgr;g each; −140 mV). Confocal microscopy analysis showed that the fluorescent protein-tagged S369X subunits were predominantly retained in the ER when expressed alone; however, the expression of S369X subunits to the plasma membrane was partially restored when coexpressed with WT. Fluorescence resonance energy transfer analysis demonstrated direct protein-protein interactions between WT and S369X subunits in the intracellular compartment. Conclusions— The S369X mutation causes a loss of the ER export motif. However, the trafficking deficiency can be partially rescued by directly assembling with the WT protein, resulting in a limited restoration of plasma membrane localization and channel function. This alleviation may explain why our patient presented with a relatively mild ATS phenotype.

Cinnamyl-3,4-dihydroxy-α-cyanocinnamate and nordihydroguaiaretic acid inhibit human Kv1.5 currents independently of lipoxygenase

In humans, Kv1.5 (hKv1.5) channels conduct the ultra-rapid delayed rectifier K(+) current (I(Kur)) that is important for the repolarization of cardiac action potentials. We aimed at examining the effect of lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC), nordihydroguaiaretic acid (NDGA), and gossypol on hKv1.5 wild-type and mutant channels heterologously expressed in Chinese hamster ovary (CHO) cells, by use of the site-directed mutagenesis and whole-cell patch-clamp method. CDC and NDGA, but not gossypol, a structurally dissimilar inhibitor, reversibly inhibited hKv1.5 current in a concentration-dependent manner with IC(50) of 5.7 microM and 16.4 microM, respectively. The blockade evoked by both drugs was voltage-dependent between -20 and +10 mV (voltage range of channel opening). Moreover, this blocking action was found to progress with time during depolarizing voltage steps with a more rapid block at higher concentrations. CDC induced slight but significant delay of the deactivation rate. However, NDGA markedly slowed the deactivation time course, resulting in a tail crossover phenomenon. The recovery time constants from current block at repolarizing potentials for CDC and NDGA were 60.9 ms and 129.7 ms, respectively. Mutation of arginine 487 to valine (R487V) in the outer pore region of the channel significantly reduced the CDC action. These results demonstrate for the first time that CDC and NDGA block hKv1.5 channels by binding to the open state of the channels, independently of their effects on lipoxygenase activity. The putative binding site for CDC appears to be related to arginine 487 located in the outer pore region.

A family of hereditary long QT syndrome caused by Q738X HERG mutation

Abstract We report a family of type 2 long QT syndrome caused by a novel single nucleotide HERG mutation (c.C2212T, p.Q738X). A female complained of syncope and ECG showed long QT, flat T wave, lack of QT shortening during sinus tachycardia and subsequent Torsade de Pointes. After implantation of ICD, she has experienced no syncope. Her father exhibited advanced atrioventricular block and persistent atrial flutter, and her daughter showed long QT with broad-based, sharp T wave. A possible cause to show wide variety of ECG phenotypes under the same mutation in this family is discussed.

Blockade of cardiac ATP-sensitive k+ channel by cibenzoline targets its pore-forming subunit

Several antiarrhythmic agents with Na-channel blocking action have been shown to inhibit cardiac K(ATP) channels. We used cibenzoline to examine its precise target site using patch-clamp techniques and receptor binding assays in guinea-pig ventricular myocytes. Exposure of myocytes to a glucose-free perfusate containing 1 mM cyanide produced a time-dependent shortening of the action potential duration (APD) in the current-clamp mode. Cibenzoline (30 microM) slowed the development of APD shortening (APD90 to approximately 91% vs. approximately 55% control 16 min after metabolic inhibition) at pHo 7.4, but not at pHo 6.4 (to approximately 60%). The pinacidil (30 microM)-induced K(ATP) currents were inhibited by cibenzoline in a pHo-dependent manner: the higher the pHo, the stronger the blocking effect of cibenzoline. The binding of [3H]-labeled cibenzoline was prevented by cibenzoline, but not by glibenclamide. Alkalinization produces a higher concentration of the uncharged form of cibenzoline, which can more easily permeate the cell membrane than the charged form. In NIH3T3 cells stably expressing Kir6.1, a putative pore-forming subunit of K(ATP) channel, cibenzoline but not glibenclamide inhibited the K conductance. Thus cibenzoline interacts with the channel pore-forming subunit of the K(ATP) channel (Kir6.2), but not the sulfonylurea receptor, from the cytosolic side after it permeates into the cell interior via the membrane lipid bilayer.