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.     

Detection of sequence variations in the adenomatous polyposis coli (APC) gene using denaturing high-performance liquid chromatography

We have evaluated the usefulness of denaturing high performance liquid chromatography (dHPLC) for scanning the adenomatous polyposis coli (APC) gene for point mutations, small deletions, and insertions. Our assay consists of 28 sets of primers to amplify the 15 exons of the APC gene. All PCR reactions were amplified simultaneously using the same reaction conditions in a 96-well format and then analyzed by dHPLC, using empirically determined optimum temperatures for partial fragment denaturation. Previously studied DNA specimens from 47 familial adenomatous polyposis (FAP) patients were analyzed by dHPLC and all mutations were correctly identified and confirmed by sequence analysis. This approach identified a single-base substitution in exon 6 and a 2-bp insertion in exon 15 that initially had not been detected by single-strand conformational polymorphism (SSCP) analysis. A novel mutation in exon 15 of the APC gene, 2065delG (codon 689) that had previously been undetected by the protein truncation test (PTT) was also identified by dHPLC. We present our validation studies of dHPLC technology for APC gene analysis in terms of sensitivity and specificity and compare it to current standard scanning technologies including PTT, SSCP, and conformational sensitive gel electrophoresis (CSGE).     

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.     

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.     

Cardiac ion channel gene mutations in Greek long QT syndrome patients

The long QT syndrome (LQTS) is an inherited cardiac arrhythmia that may lead to sudden death in the absence of structural heart disease. Mutations in the cardiac potassium and sodium channel genes can be found in approximately 70% of patients with a highly probable clinical diagnosis. In this study, we aimed to genotype and explore the yield of genetic testing of LQTS patients from Greece, for whom there are no collective published data available. We clinically evaluated and genetically screened 17 unrelated patients for mutations in theKCNQ1, KCNH2, SCN5A, KCNE1, andKCNE2 cardiac ion channel genes. Genetic testing was positive in 6 out of 8 patients with a highly probable clinical diagnosis of LQTS and negative for all the other patients. Two patients carriedKCNQ1 mutations (c.580G>C, c.1022C>T), while 4 patients carriedKCNH2 mutations (c.202T>C, c.1714G>A, c.3103delC, c.3136C>T). To the best of our knowledge, the last mentioned mutation (c.3136C>T) is novel. Moreover, 27 single-nucleotide polymorphisms (SNPs) were detected, 5 of which are novel. Our preliminary data indicate a low genetic diversity of the Greek LQTS genetic pool, and are in accordance with international data that genetic testing of the major LQTS genes is efficient in genotyping the majority of patients with a strong clinical diagnosis. Therefore, the transition of an LQTS genetic screening program from research to the diagnostic setting within our ethnic background is feasible.     

Evaluation of DHPLC in the analysis of hemophilia A

The manifestation of hemophilia A, a common hereditary bleeding disorder in humans, is caused by abnormalities in the factor VIII (FVIII) gene. A wide range of different mutations has been identified and provides the genetic basis for the extensive variability observed in the clinical phenotype. The knowledge of a specific mutation is of great interest as this may facilitate genetic counseling and prediction of the risk of anti-FVIII antibody development, the most serious complication in hemophilia A treatment to date. Due to its considerable size (7.2 kb of the coding sequence, represented by 26 exons), mutation detection in this gene represents a challenge that is only partially met by conventional screening methods such as denaturing gradient gel electrophoresis (DGGE) or single stranded conformational polymorphism (SSCP). These techniques are time consuming, require specific expertise and are limited to detection rates of 70-85%. In contrast, the recently introduced denaturing high performance liquid chromatography (dHPLC) offers a promising new method for a fast and sensitive analysis of PCR-amplified DNA fragments. To test the applicability of dHPLC in the molecular diagnosis of hemophilia A, we first assessed a cohort of 156 patients with previously identified mutations in the FVIII gene. Applying empirically determined exon-specific melting profiles, a total of 150 mutations (96.2%) were readily detected. Five mutations (3.2%) could be identified after temperatures were optimized for the specific nucleotide change. One mutation (0.6%) failed to produce a detectable heteroduplex signal. In a second series, we analyzed 27 hemophiliacs in whom the mutation was not identified after extensive DGGE and chemical mismatch cleavage (CMC) analysis. In 19 of these patients (70.4%), dHPLC facilitated the detection of the disease-associated nucleotide alterations. From these findings we conclude that the dHPLC technology is a highly sensitive method well suited to the molecular analysis of hemophilia A.     

Cardiac arrhythmia and thyroid dysfunction: A novel genetic link

Inherited Long QT Syndrome (LQTS), a cardiac arrhythmia that predisposes to the often lethal ventricular fibrillation, is commonly linked to mutations in KCNQ1. The KCNQ1 voltage-gated K⁺ channel α subunit passes ventricular myocyte K⁺ current that helps bring a timely end to each heart-beat. KCNQ1, like many K⁺ channel α subunits, is regulated by KCNE β subunits, inherited mutations in which also associate with LQTS. KCNQ1 and KCNE mutations are also associated with atrial fibrillation. It has long been known that thyroid status strongly influences cardiac function, and that thyroid dysfunction causes abnormal cardiac structure and rhythm. We recently discovered that KCNQ1 and KCNE2 form a thyroid-stimulating hormone-stimulated K⁺ channel in the thyroid that is required for normal thyroid hormone biosynthesis. Here, we review this novel genetic link between cardiac and thyroid physiology and pathology, and its potential influence upon future therapeutic strategies in cardiac and thyroid disease.     

Analysis of candidate genes for genotypic diagnosis in the long QT syndrome

Patients with the long QT syndrome (LQTS) suffer from cardiac arrhythmias that can lead to abrupt loss of consciousness and sudden death, already in young individuals. Thus, an early diagnosis of LQTS is essential for patients and their family members. So far, six genes (KCNQ1, HERG, SCN5A, ANK2, KCNE1, KCNE2) have been demonstrated to be involved in the development of LQTS. Since this syndrome is genetically heterogeneous and large-sized families are often not available for linkage analysis, alternative tools are required for a genetic diagnosis. To investigate genes with numerous exons, like KCNQ1, HERG, SCN5A and ANK2, segregation analysis of a Polish Romano-Ward family with eight members was performed as a reliable method faster than linkage analysis or direct sequencing. To test these four LQT loci, an appropriate selection of microsatellite markers covering different chromosomal regions was applied. Furthermore, two small genes KCNE1 and KCNE2 (at the LQT5 and LQT6 loci), and the SGK1 gene (encoding a kinase regulating KCNE1 and SCN5A channels) were sequenced. All six LQT loci and the SGK1 gene were excluded by these analyses, thus a different pathogenic mechanism of LQT syndromes can be presumed.     

Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.     

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.     

Characterization of recombinant mustard trypsin inhibitor 2 (MTI2) expressed in Pichia pastoris

KCNQ2 and KCNQ3 subunits belong to the six transmembrane domain K⁺ channel family and loss of function mutations are associated with benign familial neonatal convulsions. KCNE2 (MirP1) is a single transmembrane domain subunit first described to be a modulator of the HERG potassium channel in the heart. Here, we show that KCNE2 is present in brain, in areas which also express KCNQ2 and KCNQ3 channels. We demonstrate that KCNE2 associates with KCNQ2 and/or KCNQ3 subunits. In transiently transfected COS cells, KCNE2 expression produces an acceleration of deactivation kinetics of KCNQ2 and of the KCNQ2–KCNQ3 complex. Effects of two previously identified arrhythmogenic mutations of KCNE2 have also been analyzed.     

Novel Mechanisms of Trafficking Defect Caused by KCNQ1 Mutations Found in Long QT Syndrome

Long QT syndrome (LQTS) is a hereditary arrhythmia caused by mutations in genes for cardiac ion channels, including a potassium channel, KvLQT1. Inheritance of LQTS is usually autosomal-dominant, but autosomal-recessive inheritance can be observed in patients with LQTS accompanied by hearing loss. In this study, we investigated the functional alterations caused by KCNQ1 mutations, a deletion (delV595) and a frameshift (P631fs/19), which were identified in compound heterozygous state in two patients with autosomal-recessive LQTS not accompanied by hearing loss. Functional analyses showed that both mutations impaired cell surface expression due to trafficking defects. The mutations severely affected outward potassium currents without apparent dominant negative effects. It was found that delV595 impaired subunit binding, whereas P631fs/19 was retained in endoplasmic reticulum due to the newly added 19-amino acid sequence containing two retention motifs (R⁶³³GR and R⁶⁴⁶LR). This is the first report of novel mechanisms for trafficking abnormality of cardiac ion channels, providing us new insights into the molecular mechanisms of LQTS.     

Validation of dHPLC for molecular diagnosis of beta-thalassemia in Southern Italy

Beta-thalassemia, the most common hereditary anemia in the Mediterranean area, results from over 200 causative mutations in the beta-globin locus. The aim of this study was to validate a denaturing high-performance liquid chromatography (dHPLC)-based assay for postnatal and prenatal molecular diagnosis of beta-thalassemia in Southern Italy. Sixty beta-thalassemic patients, affected either by thalassemia intermedia or thalassemia major, were analyzed in a blind study. We also carried out prenatal molecular diagnosis in 12 couples at-risk for having affected offspring. Chorionic villi samples were subjected to dHPLC analysis upon molecular characterization of the parental beta-globin alleles. Direct sequence analysis was used to validate each result, showing an accuracy rate of 100% for dHPLC. Overall, our protocol was able to identify the responsible mutations in all 96 analyzed subjects (including 12 prenatals in at-risk pregnancies), detecting the eight most common mutations in Southern Italy. Three rare mutations (one of which, reported here for the first time) that standard mutation detection methods failed to reveal, were also identified. dHPLC assay proved to be a reliable, rapid, and sensitive method for detecting both common and rare mutations within the beta-globin gene. Because of this property our protocol has the potential to be implemented for mutational screening in different areas of high prevalence for beta-thalassemia.     

Molecular Cloning and Functional Expression of the Equine K+ Channel KV11.1 (Ether à Go-Go-Related/KCNH2 Gene) and the Regulatory Subunit KCNE2 from Equine Myocardium

The KCNH2 and KCNE2 genes encode the cardiac voltage-gated K⁺ channel K_V11.1 and its auxiliary β subunit KCNE2. K_V11.1 is critical for repolarization of the cardiac action potential. In humans, mutations or drug therapy affecting the K_V11.1 channel are associated with prolongation of the QT intervals on the ECG and increased risk of ventricular tachyarrhythmia and sudden cardiac death—conditions known as congenital or acquired Long QT syndrome (LQTS), respectively. In horses, sudden, unexplained deaths are a well-known problem. We sequenced the cDNA of the KCNH2 and KCNE2 genes using RACE and conventional PCR on mRNA purified from equine myocardial tissue. Equine K_V11.1 and KCNE2 cDNA had a high homology to human genes (93 and 88%, respectively). Equine and human K_V11.1 and K_V11.1/KCNE2 were expressed in Xenopus laevis oocytes and investigated by two-electrode voltage-clamp. Equine K_V11.1 currents were larger compared to human K_V11.1, and the voltage dependence of activation was shifted to more negative values with V_1/2 = -14.2±1.1 mV and -17.3±0.7, respectively. The onset of inactivation was slower for equine K_V11.1 compared to the human homolog. These differences in kinetics may account for the larger amplitude of the equine current. Furthermore, the equine K_V11.1 channel was susceptible to pharmacological block with terfenadine. The physiological importance of K_V11.1 was investigated in equine right ventricular wedge preparations. Terfenadine prolonged action potential duration and the effect was most pronounced at slow pacing. In conclusion, these findings indicate that horses could be disposed to both congenital and acquired LQTS.     

Expression and coassociation of ERG1, KCNQ1, and KCNE1 potassium channel proteins in horse heart

In dogs and in humans, potassium channels formed by ether-a-go-go-related gene 1 protein ERG1 (KCNH2) and KCNQ1 alpha-subunits, in association with KCNE beta-subunits, play a role in normal repolarization and may contribute to abnormal repolarization associated with long QT syndrome (LQTS). The molecular basis of repolarization in horse heart is unknown, although horses exhibit common cardiac arrhythmias and may receive drugs that induce LQTS. In horse heart, we have used immunoblotting and immunostaining to demonstrate the expression of ERG1, KCNQ1, KCNE1, and KCNE3 proteins and RT-PCR to detect KCNE2 message. Peptide N-glycosidase F-sensitive forms of horse ERG1 (145 kDa) and KCNQ1 (75 kDa) were detected. Both ERG1 and KCNQ1 coimmunoprecipitated with KCNE1. Cardiac action potential duration was prolonged by antagonists of either ERG1 (MK-499, cisapride) or KCNQ1/KCNE1 (chromanol 293B). Patch-clamp analysis confirmed the presence of a slow delayed rectifier current. These data suggest that repolarizing currents in horses are similar to those of other species, and that horses are therefore at risk for acquired LQTS. The data also provide unique evidence for coassociation between ERG1 and KCNE1 in cardiac tissue.     

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.     

Gating-Related Molecular Motions in the Extracellular Domain of the I<Subscript>Ks</Subscript> Channel: Implications for I<Subscript>Ks</Subscript> Channelopathy

Cardiac slow delayed rectifier (I_Ks) channel complex consists of KCNQ1 channel and KCNE1 auxiliary subunits. The extracellular juxtamembranous region of KCNE1 is an unstructured loop that contacts multiple KCNQ1 positions in a gating-state-dependent manner. Congenital arrhythmia-related mutations have been identified in the extracellular S1–S2 linker of KCNQ1. These mutations manifest abnormal phenotypes only when coexpressed with KCNE1, pointing to the importance of proper KCNQ1/KCNE1 interactions here in I_Ks channel function. We investigate the interactions between the KCNE1 loop (positions 36–47) and KCNQ1 S1–S2 linker (positions 140–148) by means of disulfide trapping and voltage clamp techniques. During transitions among the resting-state conformations, KCNE1 positions 36–43 make contacts with KCNQ1 positions 144, 145, and 147 in a parallel fashion. During conformational changes in the activated state, KCNE1 position 40 can make contacts with all three KCNQ1 positions, while the neighboring KCNE1 positions (36, 38, 39, and 41) can make contact with KCNQ1 position 147. Furthermore, KCNQ1 positions 143 and 146 are high-impact positions that cannot tolerate cysteine substitution. To maintain the proper I_Ks channel function, position 143 requires a small side chain with a hydroxyl group, and position 146 requires a negatively charged side chain. These data and the proposed molecular motions provide insights into the mechanisms by which mutations in the extracellular juxtamembranous region of the I_Ks channel impair its function.     

Regulation of the Kv2.1 Potassium Channel by MinK and MiRP1

Kv2.1 is a voltage-gated potassium (Kv) channel α-subunit expressed in mammalian heart and brain. MinK-related peptides (MiRPs), encoded by KCNE genes, are single–transmembrane domain ancillary subunits that form complexes with Kv channel α-subunits to modify their function. Mutations in human MinK (KCNE1) and MiRP1 (KCNE2) are associated with inherited and acquired forms of long QT syndrome (LQTS). Here, coimmunoprecipitations from rat heart tissue suggested that both MinK and MiRP1 form native cardiac complexes with Kv2.1. In whole-cell voltage-clamp studies of subunits expressed in CHO cells, rat MinK and MiRP1 reduced Kv2.1 current density three- and twofold, respectively; slowed Kv2.1 activation (at +60 mV) two- and threefold, respectively; and slowed Kv2.1 deactivation less than twofold. Human MinK slowed Kv2.1 activation 25%, while human MiRP1 slowed Kv2.1 activation and deactivation twofold. Inherited mutations in human MinK and MiRP1, previously associated with LQTS, were also evaluated. D76N–MinK and S74L–MinK reduced Kv2.1 current density (threefold and 40%, respectively) and slowed deactivation (60% and 80%, respectively). Compared to wild-type human MiRP1–Kv2.1 complexes, channels formed with M54T– or I57T–MiRP1 showed greatly slowed activation (tenfold and fivefold, respectively). The data broaden the potential roles of MinK and MiRP1 in cardiac physiology and support the possibility that inherited mutations in either subunit could contribute to cardiac arrhythmia by multiple mechanisms. Electronic supplementary material  The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Z. A. McCrossan and T. K. Roepke have contributed equally to this work.     

KCNE2 confers background current characteristics to the cardiac KCNQ1 potassium channel

Mutations in HERG and KCNQ1 (or KVLQT1) genes cause the life-threatening Long QT syndrome. These genes encode K(+) channel pore-forming subunits that associate with ancillary subunits from the KCNE family to underlie the two components, I(Kr) and I(Ks), of the human cardiac delayed rectifier current I(K). The KCNE family comprises at least three members. KCNE1 (IsK or MinK) recapitulates I(Ks) when associated with KCNQ1, whereas it augments the amplitude of an I(Kr)-like current when co-expressed with HERG. KCNE3 markedly changes KCNQ1 as well as HERG current properties. So far, KCNE2 (MirP1) has only been shown to modulate HERG current. Here we demonstrate the interaction of KCNE2 with the KCNQ1 subunit, which results in a drastic change of KCNQ1 current amplitude and gating properties. Furthermore, KCNE2 mutations also reveal their specific functional consequences on KCNQ1 currents. KCNQ1 and HERG appear to share unique interactions with KCNE1, 2 and 3 subunits. With the exception of KCNE3, mutations in all these partner subunits have been found to lead to an increased propensity for cardiac arrhythmias.     

Structure of KCNE1 and implications for how it modulates the KCNQ1 potassium channel

KCNE1 is a single-span membrane protein that modulates the voltage-gated potassium channel KCNQ1 (K V7.1) by slowing activation and enhancing channel conductance to generate the slow delayed rectifier current ( I Ks) that is critical for the repolarization phase of the cardiac action potential. Perturbation of channel function by inherited mutations in KCNE1 or KCNQ1 results in increased susceptibility to cardiac arrhythmias and sudden death with or without accompanying deafness. Here, we present the three-dimensional structure of KCNE1. The transmembrane domain (TMD) of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers. Experimentally restrained docking of the KCNE1 TMD to a closed state model of KCNQ1 suggests that KCNE1 slows channel activation by sitting on and restricting the movement of the S4-S5 linker that connects the voltage sensor to the pore domain. We postulate that this is an adhesive interaction that must be disrupted before the channel can be opened in response to membrane depolarization. Docking to open KCNQ1 indicates that the extracellular end of the KCNE1 TMD forms an interface with an intersubunit cleft in the channel that is associated with most known gain-of-function disease mutations. Binding of KCNE1 to this "gain-of-function cleft" may explain how it increases conductance and stabilizes the open state. These working models for the KCNE1-KCNQ1 complexes may be used to formulate testable hypotheses for the molecular bases of disease phenotypes associated with the dozens of known inherited mutations in KCNE1 and KCNQ1.