Electrophysiological substrates for atrial fibrillation: genetic predisposition and acquired condition

Im ersten Teil der Arbeit wurde eine genetische Disposition für Vorhofflimmern (VHF) untersucht. Der Einzelnukleotidpolymorphismus ("single nucleotide polymorphism", SNP) 38G/S befindet sich im N-Terminus der ß-Untereinheit KCNE1. Diese ß-Untereinheit konstituiert gemeinsam mit der alpha-Untereinheit KCNQ1 die langsame Komponente des verzögerten Gleichrichterstromes, IKs. Die ß-Untereinheit hat hierbei eine modulierende Funktion. Frühere Studien beschäftigten sich hauptsächlich mit der transmembranären Domäne und dem C-Terminus. Über die Rolle des N-Terminus war bislang wenig bekannt. Das Ziel der vorliegenden Arbeit war es, die Aufgabe des N-Terminus bei der Modulation der alpha-Untereinheit zu identifizieren. Außerdem sollte festgestellt werden, welche Aminosäuren hierbei besonders von Bedeutung sind. Zu diesem Zweck wurden diverse Konstrukte synthetisiert. Für das Konstrukt delta1-38’ wurden die Aminosäuren 1-38 und damit der Großteil des N-Terminus entfernt. Das Konstrukt "linker" enthält anstelle des Glyzins oder Serins an Position 38 fünf Alanine. In der Nähe der von dem SNP betroffenen Aminosäure befinden sich des Weiteren drei Arginine, die mit jeweils einem Alanin substituiert wurden. Für alle Versuche diente die nicht VHF-assoziierte Variante des SNPs als Kontrolle. Alle Konstrukte konnten erfolgreich heterolog exprimiert werden und gleichermaßen mit der alpha-Untereinheit immunopräzipitiert werden. Die aus der Co-Transfektion von KCNQ1 und KCNE1 resultierende Stromdichte wurde mittels "Patch-clamp"-Technik untersucht. Im Vergleich zum Kontrollstrom (KCNQ1 + KCNE1-38S) waren die Ströme aller anderen Gruppen während De- und Repolarisation signifikant kleiner. Zellfraktionierung und konfokale Mikroskopie zeigten, dass im Vergleich zur Kontrolle alle anderen Konstrukte eine verminderte Plasmamembranlokalisation aufwiesen. Die Aufgabe des N-Terminus liegt offensichtlich im Transport beider Untereinheiten an die Plasmamembran und/oder der Verankerung dort. Sowohl die Aminosäure in Position 38 als auch die drei N-terminalen Arginine in der Nähe scheinen für den hier gesuchten Mechanismus von Bedeutung zu sein. Zukünftige Experimente könnten beispielsweise 3D-Simulationen der Proteinfaltung beinhalten, um die potentielle Membranverankerung weiter zu untersuchen. Der zweite Teil der Arbeit untersuchte erworbene elektrophysiologische Veränderungen im Rahmen von VHF am Beispiel der einwärts gleichrichtenden Kaliumströme IK1 und IKACh. Es sollten die zugrunde liegenden regulatorischen Mechanismen für die Heraufregulierung von IK1 und IKACh bei VHF untersucht werden. Alle Experimente wurden an humanem Gewebe des linken Vorhofs durchgeführt. Das Gewebe stammt von VHF-Patienten, die sich einer Mitralklappen-Operation unterzogen. Als Kontrolle wurde Gewebe von Patienten im Sinusrhythmus (SR) verwendet. Zunächst wurde untersucht, ob transkriptionelle und/oder posttranskriptionelle Veränderungen oder funktionelle Effekte der Heraufregulierung der Ströme zugrunde liegen. Entsprechend wurde die Proteinexpression mittels Western Blot quantifiziert. Die Quantifizierung der mRNA erfolgte per Realtime-PCR. Veränderungen für IK1 konnten sowohl auf mRNA- als auch auf translationaler Ebene beobachtet werden. Protein- und mRNA-Expression von Kir2.1, der zugrunde liegenden Proteinuntereinheit, waren bei VHF signifikant erhöht; die Expression der inhibitorischen miR-1 war reduziert. Die Bestimmung der Protein- und mRNA-Expression der zugrunde liegenden Proteinuntereinheiten für den Strom IKACh zeigte dagegen keinen Unterschied zwischen Gewebe von Patienten mit VHF und SR. Eine funktionelle Regulierung schien daher möglich. Die Expression der IKACh modulierenden Proteine Calmodulin und G alpha i-3 unter VHF zeigte jedoch keinen signifikanten Unterschied zu der SR-Gruppe. Es war eine Tendenz zur Reduktion des inhibierenden G alpha i-3 zu beobachten. Die Regulierung von IKACh,c bei VHF bleibt in zukünftigen Arbeiten zu untersuchen. Ein möglicher Versuch wäre, therapeutisch in die Regulation der Kir-Untereinheiten einzugreifen, um das VHF-unterstützende, elektrische "Remodeling" des IK1 zu verhindern.The first part of the thesis dealt with a genetic predisposition to Atrial fibrillation (AF). The N-terminal KCNE1 polymorphism 38S is highly prevalent and KCNE1-38G is associated with AF. Together with the alpha-subunit the ß-subunit KCNE1 constitutes the slow component of the delayed rectifier current, IKs. The ß-subunit has a modulatory function. Previous studies regarding this protein mainly investigated its transmembrane domain and C-terminus. Detailed structure-function relationship of the KCNE1 N-terminus for IKs modulation is poorly understood and was subject of this study. KCNE1-constructs with N-terminal mutations disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 and constructs modifying position 38 including an N-terminal truncation mutation were studied. All constructs expressed heterogenously and on the same level. Co-immunoprecipitation with the alpha-subunit was successful for all constructs. IKs resulting from co-expression of KCNQ1 with non-AF ‘38S’ was greater than with KCNE1-38G or with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct. Western blots from plasma membrane preparations and confocal images consistently showed a greater amount of KCNQ1 protein at the plasma membrane in cells co-transfected with the non-AF ‘38S’ than with the other constructs. The results of this project indicate N-terminal position 38 and arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of IKs. This work provides evidence for a role of these N-terminal amino-acids in membrane trafficking and/or anchoring of the delayed rectifier current complex. Future tasks could involve 3D protein folding simulations in order to investigater further into membrane anchoring. The second part of the thesis investigated acquired electrophysiological changes in AF. It aimed to evaluate regulatory mechanisms underlying these changes in inward rectifier currents IK1 and constitutively active IKACh. All experiments were conducted on human left atrial tissue from patients undergoing mitral valve repair. IK1 and IKACh density was increased in cells from patients with AF. IK1 underlying subunit Kir2.1 showed increased protein and mRNA expression. Also inhibitory microRNA-1 was reduced. Protein and mRNA expression of subunits Kir3.1 and Kir3.4, underlying IKACh, was unchanged. Functional modulators of Kir3 currents Calmodulin and G alpha i-3 were quantified as well. While Calmodulin was unchanged inhibitory G alpha i-3 tended to reduction. Underlying mechanisms of IKACh upregulation needs to be further studied. A therapeutic approach to the underlying mechanisms of IK1 upregulation might inhibit the AF maintaining Remodeling of the inward rectifier current

<資料>新西蘭,加奈陀,印度の中央銀行設立計畫

<p><b>Panels A–C,</b> representative images obtained from confocal microscopy of transiently transfected HEK cells. R835Q mutant channels do not appear differently distributed in comparison to WT KCNH2. <b>D</b>, Immunoblots using anti-erg1 (2, 5 µg/mL) of crude membrane extracts from heterologous expression in HEK cells, indicating equal protein expression level. Illustrated below are endoplasmic reticulum and plasma membrane fraction with respective markers of equal protein loading (calnexin for endoplasmic reticulum, spectrin for plasma membranes). Exemplary Western blots of preparations at physiological temperature (37°C) and 40°C (to simulate febrile illness of the index patient’s brother) are shown. No differences were observed in Kv11.1-WT or Kv11.1-R835Q plasma membrane representation of the two proteins under the two conditions. ER: endoplasmic reticulum fraction; PM: plasma-membrane fraction; WT: wild type; NT: non-transfected cells.</p

N-Terminal Arginines Modulate Plasma-Membrane Localization of Kv7.1/KCNE1 Channel Complexes

BACKGROUND AND OBJECTIVE: The slow delayed rectifier current (I(Ks)) is important for cardiac action potential termination. The underlying channel is composed of Kv7.1 α-subunits and KCNE1 β-subunits. While most evidence suggests a role of KCNE1 transmembrane domain and C-terminus for the interaction, the N-terminal KCNE1 polymorphism 38G is associated with reduced I(Ks) and atrial fibrillation (a human arrhythmia). Structure-function relationship of the KCNE1 N-terminus for I(Ks) modulation is poorly understood and was subject of this study. METHODS: We studied N-terminal KCNE1 constructs disrupting structurally important positively charged amino-acids (arginines) at positions 32, 33, 36 as well as KCNE1 constructs that modify position 38 including an N-terminal truncation mutation. Experimental procedures included molecular cloning, patch-clamp recording, protein biochemistry, real-time-PCR and confocal microscopy. RESULTS: All KCNE1 constructs physically interacted with Kv7.1. I(Ks) resulting from co-expression of Kv7.1 with non-atrial fibrillation '38S' was greater than with any other construct. Ionic currents resulting from co-transfection of a KCNE1 mutant with arginine substitutions ('38G-3xA') were comparable to currents evoked from cells transfected with an N-terminally truncated KCNE1-construct ('Δ1-38'). Western-blots from plasma-membrane preparations and confocal images consistently showed a greater amount of Kv7.1 protein at the plasma-membrane in cells co-transfected with the non-atrial fibrillation KCNE1-38S than with any other construct. CONCLUSIONS: The results of our study indicate that N-terminal arginines in positions 32, 33, 36 of KCNE1 are important for reconstitution of I(Ks). Furthermore, our results hint towards a role of these N-terminal amino-acids in membrane representation of the delayed rectifier channel complex

mRNA expression levels.

<p>Mean±SEM results of quantitative real-time PCR for mRNA expression of KCNE1 (<b>A</b>) and Kv7.1 (<b>B</b>) from HEK cells transfected with Kv7.1 and the various KCNE1 constructs (n = 5 transfections for each panel). Data were normalized to ß-actin expression. There were no differences in mRNA transcription that could account for changes in membrane currents.</p

Biophysical characteristics.

<p><b>A</b>, illustrates half-activation voltages (V₅₀) of currents resulting from expression of Kv7.1 with respective constructs. Mean±SEM V₅₀ values were similar between constructs ‘38S’: 5.8±3.7 mV, ‘38G’: −2.7±4.1 mV, ‘Δ1-38’: 6.0±4.1 mV, ‘linker’: 1.0±2.5 mV, ‘38S-3xA’: 3.4±8.6 mV, ‘38G-3xA’: 8.7±6.8 mV; <i>P</i> = n.s vs. ‘38S’. Lines shown are Boltzmann fits to mean data (obtained with the formula: A = A₀/(1+exp[(V₅₀-V)/S])). <b>B</b> shows results of mono-exponential fits (y = A^(−t/τ)+C) to activating currents with time-constants plotted over a test potential of 0 mV. <b>C</b> shows results of mono-exponential fits to deactivating ionic currents. Currents obtained from co-transfection of Kv7.1 with ‘linker’ and ‘38S-3xA’ deactivated more slowly than currents obtained with the remainder of the constructs (<i>P</i><0.05 vs. ‘38S’). TP – test potential.</p

Primers for KCNE1 mutagenesis.

<p>Mutation containing sequences in italic, underlined.</p

Primers for quantitation of Kv7.1 and KCNE1 mRNA.

<p>F – forward primer, R – reverse primer.</p

Schematic of KCNE1 and constructs.

<p>This figure schematically illustrates KCNE1 structure and mutants used in the present study. <b>A, left</b>, alignment of KCNE1 sequences from various mammalian species. Grey underlines conserved residues. Glycine at position 38 is not strongly conserved among species providing no first-glance evidence for evolutionary importance. <b>A, right</b>, schematic of KCNE1 at the membrane with the N-terminal part oriented towards the cell exterior and C-terminus towards the cytosol. <b>B</b>, schematic of KCNE1 N-terminal constructs and mutations created for the present study. Ten N-terminal amino-acids (AA) illustrate differences between KCNE1 constructs. Position 38 carries a glycine in the wild-type (common allele) and is associated with atrial fibrillation. Position 38 carries a serine in the prevalent single nucleotide polymorphism. One of the constructs contained an N-terminal truncation (‘Δ1-38’), another one (‘linker’) replaced position 38 by 5 alanines. Additionally, three positively-charged arginines at positions 32, 33 and 36 have been exchanged for alanines in order to probe the role of these AA in KCNE1 function.</p

Immunodetection of heterologously expressed constructs.

<p>Immunodetection of flag-tagged KCNE1 constructs. <b>A</b>, crude membrane preparations from HEK cells transiently transfected with respective KCNE1 constructs. Actin (∼42 kD) is shown as loading control. <b>B</b>, effective co-immunoprecipitation (IP) occurred for Kv7.1 with all flag-tagged KCNE1 constructs. The upper blot shows protein samples from HEK cells precipitated by anti-flag and bands detected by anti-Kv7.1. The lower blot shows samples precipitated by anti-Kv7.1 and bands detected by anti-flag antibody (n = 2 experiments each). <b>C</b> shows respective confocal images of KCNE1 subunits expressed without Kv7.1. Bars represent 5 µm. Images are representative of at least 5 different experiments. NT – non-transfected control, IP – immunoprecipitation.</p