Trafficking defect and proteasomal degradation contribute to the phenotype of a novel KCNH2 long QT syndrome mutation

The Kv11.1 (hERG) K+ channel plays a fundamental role in cardiac repolarization. Missense mutations in KCNH2, the gene encoding Kv11.1, cause long QT syndrome (LQTS) and frequently cause channel trafficking-deficiencies. This study characterized the properties of a novel KCNH2 mutation discovered in a LQT2 patient resuscitated from a ventricular fibrillation arrest. Proband genotyping was performed by SSCP and DNA sequencing. The electrophysiological and biochemical properties of the mutant channel were investigated after expression in HEK293 cells. The proband manifested a QTc of 554 ms prior to electrolyte normalization. Mutation analysis revealed an autosomal dominant frameshift mutation at proline 1086 (P1086fs+32X; 3256InsG). Co-immunoprecipitation demonstrated that wild-type Kv11.1 and mutant channels coassemble. Western blot showed that the mutation did not produce mature complex-glycosylated Kv11.1 channels and coexpression resulted in reduced channel maturation. Electrophysiological recordings revealed mutant channel peak currents to be similar to untransfected cells. Co-expression of channels in a 1∶1 ratio demonstrated dominant negative suppression of peak Kv11.1 currents. Immunocytochemistry confirmed that mutant channels were not present at the plasma membrane. Mutant channel trafficking rescue was attempted by incubation at reduced temperature or with the pharmacological agents E-4031. These treatments did not significantly increase peak mutant currents or induce the formation of mature complex-glycosylated channels. The proteasomal inhibitor lactacystin increased the protein levels of the mutant channels demonstrating proteasomal degradation, but failed to induce mutant Kv11.1 protein trafficking. Our study demonstrates a novel dominant-negative Kv11.1 mutation, which results in degraded non-functional channels leading to a LQT2 phenotype.     

Biophysical properties of mutant KCNQ1 S277L channels linked to hereditary long QT syndrome with phenotypic variability

Hereditary long QT syndrome (LQTS) is associated with ventricular torsade de pointes tachyarrhythmias and sudden cardiac death. Mutations in a cardiac voltage-gated potassium channel, KCNQ1, induce the most frequent variant of LQTS. We identified a KCNQ1 missense mutation, KCNQ1 S277L, in a patient presenting with recurrent syncope triggered by emotional stress (QTc = 528 ms). This mutation is located in the conserved S5 transmembrane region of the KCNQ1 channel. Using in vitro electrophysiological testing in the Xenopus oocyte expression system, the S277L mutation was found to be non-functional and to suppress wild type currents in dominant-negative fashion in the presence and in the absence of the regulatory ß-subunit, KCNE1. In addition, expression of S277L and wild type KCNQ1 with KCNE1 resulted in a shift of the voltage-dependence of activation by − 8.7 mV compared to wild type I_Ks, indicating co-assembly of mutant and wild type subunits. The electrophysiological phenotype corresponds well with the severe clinical phenotype of the index patient. However, investigation of family members revealed three patients that exhibit asymptomatic QT interval prolongation (QTc = 493-518 ms). In conclusion, this study emphasizes the value of biophysical testing to provide mechanistic evidence for pathogenicity of ion channel mutations identified in LQTS patients. The inconsistent association of the KCNQ1 S277L mutation with the clinical presentation suggests that additional genetic, epigenetic, or environmental factors play a role in defining the individual clinical LQTS phenotype.     

Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome

KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.     

Biophysical characteristics of a new mutation on the KCNQ1 potassium channel (L251P) causing long QT syndrome

The congenital long QT syndrome (LQTS) is a hereditary cardiac disease characterized by prolonged ventricular repolarization, syncope, and sudden death. Mutations causing LQTS have been identified in various genes that encode for ionic channels or their regulatory subunits. Several of these mutations have been reported on the KCNQ1 gene encoding for a potassium channel or its regulatory subunit (KCNE1). In this study, we report the biophysical characteristics of a new mutation (L251P) in the transmembrane segment 5 (S5) of the KCNQ1 potassium channel. Potassium currents were recorded from CHO cells transfected with either wild type or mutant KCNQ1 in the presence or in the absence of its regulatory subunit (KCNE1), using the whole-cell configuration of the patch clamp technique. Wild-type KCNQ1 current amplitudes are increased particularly by KCNE1 co-expression but no current is observed with the KCNQ1 (L251P) mutant either in the presence or in the absence of KCNE1. Coexpressing KCNE1 with equal amount of cDNAs encoding wild type and mutant KCNQ1 results in an 11-fold reduction in the amplitude of potassium currents. The kinetics of activation and inactivation and the activation curve are minimally affected by this mutation. Our results suggest that the dominant negative effect of the P251L mutation on KCNQ1 channel explains the prolonged repolarization in patients carrying this mutation.     

Buprenorphine as a safe alternative to methadone in a patient with acquired long QT syndrome: a case report

A 52-year-old man with a medical history of intravenous drug abuse was admitted to our hospital with syncope due to torsades de pointes (TdP). Two days earlier, he had used methadone. The electrocardiogram showed a prolonged corrected QT interval (QTc) of 600 ms. Continuous telemetry observation showed multiple episodes of TdP. The patient was diagnosed with bradyarrhythmia-induced TdP with acquired long QT syndrome resulting from methadone use. The QTc normalised within 2 weeks after discontinuation of the methadone. In this case of a patient with opioid dependency, there is a reasonable risk of repeated methadone use. Therefore, implantable cardioverter defibrillator or pacemaker implantation is justified but risky because of possible infections when using intravenous drugs. Given the high mortality rates seen in untreated illicit opioid users, this patient needs an alternative pharmacological treatment. Buprenorphine is an opiate-receptor agonist associated with less QTc prolongation. The patient was referred to a rehab clinic and treated with an oral combination of buprenorphine and naloxone (Suboxone). During this therapy, his QTc remained normal.     

Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long‐QT syndrome

Patient‐specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long‐QT syndromes (LQTS). To study the LQT2‐associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (I_Kr) conducted by the HERG channel and the action potential (AP) duration in iPSC‐derived cardiomyocytes (CMs). Introduction of the same mutation reduced I_Kr and prolonged the AP duration in hESC‐derived CMs. Further characterization of N996I‐HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.     

Gene sequencing in neonates and infants with the long QT syndrome

The objective was to analyze the clinical and molecular findings in a cohort of neonates and infants with the autosomal dominant long QT syndrome (LQTS). Those affected face a high risk of ventricular arrhythmia resulting in syncope, seizure or sudden death. Blood samples submitted for molecular diagnostic studies on 7 infants were subject to DNA extraction and mutation analysis of 18 selected exons in 5 LQTS genes (KCNQ1, HERG, SCN5A, KCNE1, and KCNE2). We detected 11 mutations in these 7 patients. Four patients had 2 mutations in 1 gene (compound heterozygotes) or 2 different genes (digenic inheritance), while 3 patients had 1 mutation each. Except for 1 mutation in KCNE1, all other mutations were detected alone or in combination within HERG and the SCN5A genes. Four of the mutations we found are novel. The lethal nature of the LQTS demands careful attention to the family history and prompt and precise diagnosis and treatment with serious consideration of endocardial pacemaker implantation. While much larger studies are needed, our data suggest that compound heterozygotes or those with 2 mutations in different genes are likely to have a more severe LQTS including early manifestations in neonates and infants.     

Identification and characterisation of a novel KCNQ1 mutation in a family with Romano-Ward syndrome

Romano-Ward syndrome (RWS), the autosomal dominant form of the congenital long QT syndrome, is characterised by prolongation of the cardiac repolarisation process associated with ventricular tachyarrhythmias of the torsades de pointes type. Genetic studies have identified mutations in six ion channel genes, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 and the accessory protein Ankyrin-B gene, to be responsible for this disorder. Single-strand conformation polymorphism (SSCP) analysis and subsequent DNA sequence analysis have identified a KCNQ1 mutation in a family that were clinically conspicuous due to several syncopes and prolonged QTc intervals in the ECG. The mutant subunit was expressed and functionally characterised in the Xenopus oocyte expression system. A novel heterozygous missense mutation with a C to T transition at the first position of codon 343 (CCA) of the KCNQ1 gene was identified in three concerned family members (QTc intervals: 500, 510 and 530 ms, respectively). As a result, proline 343 localised within the highly conserved transmembrane segment S6 of the KCNQ1 channel is replaced by a serine. Co-expression of mutant (KCNQ1-P343S) and wild-type (KCNQ1) cRNA in Xenopus oocytes produced potassium currents reduced by approximately 92%, while IKs reconstitution experiments with a combination of KCNQ1 mutant, wild-type and KCNE1 subunits yielded currents reduced by approximately 60%. A novel mutation (P343S) identified in the KCNQ1 subunit gene of three members of a RWS family showed a dominant-negative effect on native IKs currents leading to prolongation of the heart repolarisation and possibly increases the risk of malign arrhythmias with sudden cardiac death.     

An electrifying iPSC disease model: long QT syndrome type 2 and heart cells in a dish

In a recent Nature paper, Itzhaki et?al. (2011) generate induced pluripotent stem cells (iPSCs) from patients with a potentially fatal inherited arrhythmia, long QT syndrome type 2. Cardiomyocytes differentiated from the patient-derived iPSCs exhibit the hallmark cardiac electrical signatures of the disease, which can be reversed by pharmacological intervention.     

In vivo gene transfer of Kv1.5 normalizes action potential duration and shortens QT interval in mice with long QT phenotype

Mutations in cardiac voltage-gated K+ channels cause long QT syndrome (LQTS) and sudden death. We created a transgenic mouse with a long QT phenotype (Kv1DN) by overexpression of a truncated K+ channel in the heart and investigated whether the dominant negative effect of the transgene would be overcome by the direct injection of adenoviral vectors expressing wild-type Kv1.5 (AV-Kv1.5) into the myocardium. End points at 3-10 days included electrophysiology in isolated cardiomyocytes, surface ECG, programmed stimulation of the right ventricle, and in vivo optical mapping of action potentials and repolarization gradients in Langendorff-perfused hearts. Overexpression of Kv1.5 reconstituted a 4-aminopyridine-sensitive outward K+ current, shortened the action potential duration, eliminated early afterdepolarizations, shortened the QT interval, decreased dispersion of repolarization, and increased the heart rate. Each of these changes is consistent with a physiologically significant primary effect of adenoviral expression of Kv1.5 on ventricular repolarization of Kv1DN mice.     

Preparation, functional characterization, and NMR studies of human KCNE1, a voltage-gated potassium channel accessory subunit associated with deafness and long QT syndrome

KCNE1, also known as minK, is a member of the KCNE family of membrane proteins that modulate the function of KCNQ1 and certain other voltage-gated potassium channels (KV). Mutations in human KCNE1 cause congenital deafness and congenital long QT syndrome, an inherited predisposition to potentially life-threatening cardiac arrhythmias. Although its modulation of KCNQ1 function has been extensively characterized, many questions remain regarding KCNE1's structure and location within the channel complex. In this study, KCNE1 was overexpressed in Escherichia coli and purified. Micellar solutions of the protein were then microinjected into Xenopus oocytes expressing KCNQ1 channels, followed by electrophysiological recordings aimed at testing whether recombinant KCNE1 can co-assemble with the channel. Nativelike modulation of channel properties was observed following injection of KCNE1 in lyso-myristoylphosphatidylglycerol (LMPG) micelles, indicating that KCNE1 is not irreversibly misfolded and that LMPG is able to act as a vehicle for delivering membrane proteins into the membranes of viable cells. 1H-15N TROSY NMR experiments indicated that LMPG micelles are well-suited for structural studies of KCNE1, leading to assignment of its backbone resonances and to relaxation studies. The chemical shift data confirmed that KCNE1's secondary structure includes several alpha-helices and demonstrated that its distal C-terminus is disordered. Surprisingly, for KCNE1 in LMPG micelles, there appears to be a break in alpha-helicity at sites 59-61, near the middle of the transmembrane segment, a feature that is accompanied by increased local backbone mobility. Given that this segment overlaps with sites 57-59, which are known to play a critical role in modulating KCNQ1 channel activation kinetics, this unusual structural feature likely has considerable functional relevance.     

Genomic organization and mutational analysis of KVLQT1, a gene responsible for familial long QT syndrome

To elucidate the role of the KVLQT1 gene in the pathogenesis of long QT syndrome (LQTS), we have established a screening system for detecting KVLQT1 mutations by the polymerase chain reaction-single strand conformation polymorphism technique (PCR-SSCP). We first determined exon/intron boundaries and flanking intronic sequences, and found that the KVLQT1 gene consists of 17 coding exons. Subsequently, we synthesized oligonucleotide primers to cover the coding region and the flanking intronic sequences, and searched for mutations in 31 Japanese LQTS families. When genomic DNA from each proband was examined by PCR-SSCP followed by direct DNA sequencing, mutations were detected in five families; two independent families carried the same mutation and three of the four were novel. Each mutation was present in affected relatives of the respective proband. This work will enable us to search more thoroughly for LQTS mutations associated with KVLQT1, and eventually will help us in finding genotype/phenotype relationships. Received: 20 March 1998 / Accepted: 30 April 1998     

Genotype and clinical characteristics of congenital long QT syndrome in Thailand

Background

Congenital long QT syndrome (LQTS) is an inheritable arrhythmic disorder which is linked to at least 17 genes. The clinical characteristics and genetic mutations may be variable among different population groups and they have not yet been studied in Thai population.

Mutation analysis of candidate genes SCN1B, KCND3 and ANK2 in patients with clinical diagnosis of long QT syndrome

The long QT syndrome (LQTS) is a monogenic disorder characterized by prolongation of the QT interval on electrocardiogram and syncope or sudden death caused by polymorphic ventricular tachycardia (torsades de pointes). In general, mutations in cardiac ion channel genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) have been identified as a cause for LQTS. About 50-60 % of LQTS patients have an identifiable LQTS causing mutation in one of mentioned genes. In a group of 12 LQTS patients with no identified mutations in these genes we have tested a hypothesis that other candidate genes could be involved in LQTS pathophysiology. SCN1B and KCND3 genes encode ion channel proteins, ANK2 gene encodes cytoskeletal protein interacting with ion channels. To screen coding regions of genes SCN1B, KCND3, and 10 exons of ANK2 following methods were used: PCR, SSCP, and DNA sequencing. Five polymorphisms were found in screened candidate genes, 2 polymorphisms in KCND3 and 3 in SCN1B. None of found polymorphisms has coding effect nor is located close to splice sites or has any similarity to known splicing enhancer motifs. Polymorphism G246T in SCN1B is a novel one. No mutation directly causing LQTS was found. Molecular mechanism of LQTS genesis in these patients remains unclear.     

Mutation analysis in congenital Long QT Syndrome--a case with missense mutations in KCNQ1 and SCN5A

Long QT Syndrome (LQTS) is a cardiac disease characterized by a prolonged QT interval on a surface electrocardiogram (ECG) and by clinical symptoms such as seizures, syncope, and cardiac sudden death. At present, causal mutations of LQTS have been identified in five cardiac ion-channel genes. Because a causal mutation is usually unique to a specific family and can be located in any region of any of these five genes, a mutation analysis effort may require screening of the complete coding regions of each of these genes. The causative nature of a detected mutation can then be determined either by family history or by functional studies, such as the electrophysiological signature of the mutation. Here we describe a mutation analysis of an LQTS patient who carries two heterozygous missense mutations in two different LQTS genes. The first mutation identified, A572D in SCN5A, was not linked with clinical LQTS features in the two other mutation carriers in the family; neither was it identified in 90 healthy controls. Therefore, this mutation most likely has either a mild effect on cardiac ion-channel function or represents a very rare polymorphism. The second mutation, V254M in KCNQ1, co-segregated with higher QT intervals and symptoms in other family members, and was previously reported in another LQTS family. Because the clinical LQTS symptoms are most pronounced in the proband, a combined effect of both mutations cannot be excluded, although no functional data are available to support such an hypothesis. We conclude that, for newly presented LQTS cases, a mutation analysis strategy should routinely screen the complete coding region of all LQTS genes, followed by an evaluation of the identified mutation(s) in conjunction with family or functional data.