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The KCNE1 and KCNE3 type I transmembrane-spanning β-subunits assemble with the KCNQ1 voltage-gated K+ channel to afford membrane-embedded complexes with dramatically different properties. Assembly with KCNE1 produces the very slowly activating and deactivating IKs current that shapes the repolarization phase of cardiac action potentials. Genetic mutations in KCNQ1 or KCNE1 that reduce IKs current cause long QT syndrome and predispose affected individuals to potentially fatal cardiac arrhythmias. In contrast, complexes formed between KCNQ1 and KCNE3 produce rapidly activating and mostly voltage-independent currents, properties that are essential for function in K+ recycling and Cl−secretion in gastrointestinal epithelia. This thesis addresses how these two homologous accessory peptides impart their distinctive effects on KCNQ1 channel gating by examining two important protein regions: 1) a conserved C-terminal motif in the β-subunits themselves, and 2) the voltage sensing domain of KCNQ1 channels. Sequences in both the transmembrane domain and C-terminus of KCNE1 and KCNE3 have been identified as contributing to the divergent modulatory effects that these β-subunits exert. The homology of transmembrane-abutting C-terminal residues within the KCNE family and the presence of long QT-causing mutations in this region highlight its importance. A bipartite model of modulation was proposed that suggests the transmembrane domain of KCNE1 is passive, allowing the C-terminal domain to control modulation. Chapter II builds on this model by investigating the effect of mutating specific amino acids in the KCNE1 C-terminal domain. Point mutants that produce ‘high impact’ perturbations in gating were shown to cluster in a periodic fashion, suggesting an alpha-helical secondary structure that is kinked by a conserved proline residue and interacts with the Q1 channel complex. In Chapter III, the voltage sensing domain of Q1 channels is examined in the presence of either KCNE1 or KCNE3. To determine the influence of these two peptides on voltage sensing, the position of the S4 voltage sensor was monitored using cysteine accessibility experiments. In the slowly opening KCNQ1/KCNE1 complexes, voltage sensor activation appears to occur much faster than the onset of current, suggesting that slow channel activation is not due to slowly moving voltage sensors. KCNE3, on the other hand, shifts the voltage sensor equilibrium to favor the active state, producing open channels even at negative voltages. Taken together, these findings provide mechanistic detail to illustrate how two homologous peptides radically alter the gating properties of the same K+ channel and present a structural scaffold to map protein-protein interactions.

This diploma thesis deals with study of the rate-dependence of the magnitude of a current through the heart channel that conducts slowly activating component of delayed rectifier outward current (IKs). This property is very important for the IKs channel function. When other repolarizing currents are insufficient, but also when the heart rate accelerates, especially during elevated sympathetic tone, IKs provides so-called repolarization reserve, which prevents excessive lengthening of cardiac action potential repolarization. The IKs channel structure is encoded by the KCNQ1 (pore-forming -subunit) and KCNE1 (modulatory -subunit) genes. Mutations in these genes disrupt the physiological function of the IKs channel and cause inherited arrhythmogenic syndromes, especially long QT syndrome (LQTS). Such mutations include the c.926C>T (p.T309I) mutation in the KCNQ1 gene, which results in LQTS type 1 in heterozygous carriers. The theoretical part of the thesis provides basic information about the IKs channel and the patch clamp technique, this knowledge is necessary for the practical part. The experimental part is focused on cultivation of the CHO cell line and its transient transfection for subsequent electrophysiological measurements by whole-cell patch clamp technique to study the dependence of the IKs magnitude on stimulation frequency, both in the wild type channels (i.e. without mutation) and in those with cotransfected wild type and T309I subunits.

KCNE peptides are a class of type I transmembrane ß-subunits that assemble with and modulate the gating and ion conducting properties of a variety of voltage-gated K+ channels. Accordingly, mutations that affect the assembly and trafficking of K+ channel/KCNE complexes give rise to disease. The cellular mechanisms that oversee KCNE peptide assembly with voltage-gated K+ channels have yet to be elucidated. In Chapter II, we show that KCNE1 peptides are retained in the early stages of the secretory pathway until they co-assemble with KCNQ1 K+ channel subunits. Co-assembly with KCNQ1 channel subunits mediates efficient forward trafficking of KCNE1 peptides through the biosynthetic pathway and results in cell surface expression. KCNE1 peptides possess two N-linked glycosylation sites on their extracellular N-termini. Progression of KCNE1 peptides through the secretory pathway can be visualized through maturation of N-glycans attached to KCNE1. In Chapter III, we examine the kinetics and efficiency of N-linked glycan addition to KCNE1 peptides. Mutations that prevent glycosylation of KCNE1 give rise to the disorders of arrhythmia and deafness. We show that KCNE1 acquires N-glycans co- and post-translationally. Mutations that prevent N-glycosylation at the co-translational site have a long range effect on the disruption of post-translational glycosylation and suggest a novel biogenic mechanism for disease. In Chapter IV, we determine the presence of an additional post-translational modification on KCNE1 peptides. We define specific residues as sites of attachment of this modification identified as sialylated O-glycans and show that it occurs in native cardiac tissues where KCNE1 plays a role in the maintenance of cardiac rhythm. Taken together, these observations demonstrate the importance of having correctly assembled K+ channel/KCNE complexes at the cell surface for their proper physiological function and define a role for the posttranslational modifications of KCNE peptides in the proper assembly and trafficking of K+ channel/KCNE complexes.

KCNQ1 is a homotetrameric voltage-gated potassium channel expressed in cardiomyocytes and epithelial tissues. However, currents arising from KCNQ1 have never been physiologically observed. KCNQ1 is able to provide the diverse potassium conductances required by these distinct cell types through coassembly with and modulation by type I transmembrane β-subunits of the KCNE gene family. KCNQ1-KCNE K+ channels play important physiological roles. In cardiac tissues the association of KCNQ1 with KCNE1 gives rise to IKs, the slow delayed outwardly rectifying potassium current. IKs is in part responsible for repolarizing heart muscle, and is therefore crucial in maintaining normal heart rhymicity. IKschannels help terminate each action potential and provide cardiac repolarization reserve. As such, mutations in either subunit can lead to Romano-Ward Syndrome or Jervell and Lange-Nielsen Syndrome, two forms of Q-T prolongation. In epithelial cells, KCNQ1-KCNE1, KCNQ1-KCNE2 and KCNQ1-KCNE3 give rise to potassium currents required for potassium recycling and secretion. These functions arise because the biophysical properties of KCNQ1 are always dramatically altered by KCNE co-expression. We wanted to understand how KCNE peptides are able to modulate KCNQ1. In Chapter II, we produce partial truncations of KCNE3 and demonstrate the transmembrane domain is necessary and sufficient for both assembly with and modulation of KCNQ1. Comparing these results with published results obtained from chimeric KCNE peptides and partial deletion mutants of KCNE1, we propose a bipartite modulation residing in KCNE peptides. Transmembrane modulation is either active (KCNE3) or permissive (KCNE1). Active transmembrane KCNE modulation masks juxtamembranous C-terminal modulation of KCNQ1, while permissive modulation allows C-terminal modulation of KCNQ1 to express. We test our hypothesis, and demonstrate C-terminal Long QT point mutants in KCNE1 can be masked by active trasnsmembrane modulation. Having confirmed the importance the C-terminus of KCNE1, we continue with two projects designed to elucidate KCNE1 C-terminal structure. In Chapter III we conduct an alanine-perturbation scan within the C-terminus. C-terminal KCNE1 alanine point mutations result in changes in the free energy for the KCNQ1-KCNE1 channel complex. High-impact point mutants cluster in an arrangement consistent with an alphahelical secondary structure, "kinked" by a single proline residue. In Chapter IV, we use oxidant-mediated disulfide bond formation between non-native cysteine residues to demonstrate amino acid side chains residing within the C-terminal domain of KCNE1 are close and juxtaposed to amino acid side chains on the cytoplasmic face of the KCNQ1 pore domain. Many of the amino acids identified as high impact through alanine perturbation correspond with residues identified as able to form disulfide bonds with KCNQ1. Taken together, we demonstrate that the interaction between the C-terminus of KCNE1 and the pore domain of KCNQ1 is required for the proper modulation of KCNQ1 by KCNE1, and by extension, normal IKs function and heart rhymicity.

Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d'ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d'une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l'analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n'est pas possible de s'affranchir du séquençage de type Sanger afin d'explorer les séquences d'intérêts qui n'ont pu être optimisées avec l'une ou l'autre des méthodes à la fois sur les prélèvements congelés et FFIP. L'arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine.

Background: We aimed to increase the knowledge regarding the familial arrhythmogenic disorder Long QT Syndrome (LQTS) and its recessive variant Jervell and Lange-Nielsen Syndrome (JLNS) in Sweden, including prevalences and clinical phenotypes. A specific focus was directed towards two KCNQ1 mutations –p.Y111C and p.R518X- commonly identified in Swedish LQTS index cases. Methods: Cases and families with LQTS (p.Y111C or p.R518X) and JLNS were recruited via regional clinical practices, national referrals to the Clinical Genetics laboratory, Umeå University Hospital, and a national inventory. Molecular genetics methods were used for case ascertainment. Clinical data was obtained via medical records, a questionnaire, and/or an interview. Electrocardiograms were manually assessed. In p.R518X heterozygotes intra-familial phenotypic variability (QTc and cardiac events) was assessed by analysis of sequence variants (modifier genes). The origins of the mutations p.Y111C and p.R518X were investigated using genealogical and haplotype analysis (microsatellite markers). In families sharing a common haplotype mutation age and associated prevalence was analyzed using ESTIAGE and DMLE computer software. Results: We identified p.Y111C (170 mutation-carriers) and p.R518X (101 mutation-carriers) as two major causes of LQTS/JLNS in Sweden. LQTS phenotype was revealed to be relatively benign in p.Y111C and p.R518X (annual incidence of life-threatening cardiac events, before therapy 0.05% and 0.04%, respectively). Gender-specific effects of genetic modifiers on phenotypic expression were seen. A founder origin, approximately 600-700 years ago in two northern river valleys was established for p.Y111C and p.R518X, and a high prevalence of LQTS founder descendants suggested. A minimum JLNS prevalence of 1:200 000 in preadolescent Swedish children was revealed. JLNS phenotype was mainly severe, with a cumulative incidence of life-threatening cardiac events of 53% (annual incidence rate before therapy 5%) and four sudden deaths. Possible founder effects regarding four KCNQ1 mutations; p.Y111C (8%), p.R518X (50%), c.572_576del (17%) and p.Q530X (8%) together explained 83% of the JLNS mutation-spectrum in Sweden, consisting of 8 KCNQ1 mutations. Conclusion: The high prevalence of p.Y111C- and p.R518X-related LQTS as well as JLNS revealed in Sweden could be explained by the combination of mild clinical phenotypes in heterozygotes and strong founder effects present during the population development of northern Sweden. Increased knowledge regarding the occurrence of LQTS and JLNS as well as mutation- and/or genotype-specific data constitute prerequisites for possible improvement of patient management.