Aufklärung des molekularen krankheitsauslösenden Mechanismus der progressiven kardialen Reizleitungsstörung durch die Mutation G88R im Tandem-Poren-Kanal TASK-4

Mutationen in Zwei-Porendomänen Kalium (K2P) -Kanälen konnten bereits mit verschiedenen Erkrankungen in Verbindung gebracht werden. Dazu zählt beispielsweise die dominant-negative Mutation im TRESK-Kanal (KCNK18), welche mit einer Form von familiärer Migräne mit Aura assoziiert ist (Lafrenière et al...

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Bibliographic Details
Main Author: Leonhardt, Julia
Contributors: Decher, Niels (Prof. Dr.) (Thesis advisor)
Format: Doctoral Thesis
Language:German
Published: Philipps-Universität Marburg 2021
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A variety of mutations in two-pore domain potassium (K2P)- channels have been linked to various diseases. Examples include the dominant-negative mutation in the TRESK channel (KCNK18),which is associated with a form of familial migraine with aura (Lafrenière et al., 2010) and a mutation in the TASK-3 channel (KCNK9) leading to Birk Barel syndrome with mental retardation (Barel et al., 2008). Hereditary pulmonary hypertension can be caused by various loss of function mutations in the TASK-1 channel (KCNK3) (Ma et al., 2013). A tachycardia of the right ventricular outflow tract (RVOT) can be caused by a heterozygous point mutation in the selectivity filter sequence of the TREK-1 channel (KCNK2), which leads to an increased sodium permeability and stretch-sensibility (Decher et al., 2017). In 2014, Friedrich et al. identified a novel mutation in the pH-sensitive K2P -channel TASK-4 (KCNK17) in a patient with a progressive cardiac conduction disorder (PCCD) in combination with idiopathic ventricular fibrillation. This TASK-4G88R mutation causes a significant gain of function. Interestingly, in the same patient, an additional mutation in the predisposing SCN5A gene was identified, which codes for the voltage-dependent sodium channel Nav1.5. Probst et al. (2003) previously described a disease-causing mutation affecting the same splice site, which resulted in an exon skipping, in several members of a French family with an autosomal-dominant cardiac conduction disorder. Data from "whole cell" patch-clamp measurements showed a loss of function of the Nav1.5-channel leading to a lengthening of conduction times. Therefore, the combination of the loss of function mutation in the Nav1.5 channel and the increased cellular hyperpolarization due to the TASK-4G88R mutation in the same patient may exert additive effects slowing down cardiac conductivity. This could explain the unusual phenotype of ventricular fibrillation and PCCD (Friedrich et al., 2014). The molecular mechanism underlying the altered channel function of TASK-4G88R has not been clarified yet. The main focus of this thesis was to further investigate this mechanism. For this purpose, electrophysiological experiments were carried out on Xenopus laevis oocytes using the two-electrode voltage clamp. Measurements of the ion currents after systematic introduction of all 20 different natural amino acids at position 88 in the TASK-4 protein by position-directed mutagenesis showed that normal channel function is probably mediated by the hydrophobicity of the amino acid residue located at this position. Another important focus was the question of whether the pH-sensitivity of TASK-4 is influenced by the amino acid at position 88. Based on previous electrophysiological investigations by Niemeyer et al. (2007), who postulated, that a lysine residue at position 242, located in the immediate vicinity of the channel pore, could act as the pH sensor of TASK-4. A neutralization of K242 resulted in increased current amplitudes at a neutral pH-value compared to the TASK-4WT. G88 is located directly above the selectivity filter, in the immediate vicinity of the pH sensor. In this study, I provide evidence, that the lysine residue at position 242 mediates the pH-sensitivity, but this is not related to the additional gain of function mechanism due to TASK-4G88R mutation. This could be shown by double mutations in which both the amino acid at position 242 and at position 88 were exchanged. Investigations on single-channel level using patch clamp showed an increased single-channel conductance of TASK-4G88R. Finally, I hypothesize a complex interaction of the hydrophilic arginine residue at position 88 with amino acids of the selectivity filter nearby. Such an interaction could achieve a strong electrostatic effect through the formation of a "hydrophilic cloud" and thus regulate the ion permeation in a targeted manner.