Die Bedeutung von KCNJ 15 / KCNJ 16 -Interaktionen bei einer neuartigen komplexen tubulären Nierenerkrankung
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Philipps-Universität Marburg
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Abstract
In recent decades, rare diseases have been researched that are caused by dysfunction
of ion channels in the tubular cells of the kidney and are associated with renal salt loss.
In particular, the EAST/SeSAME syndrome has been investigated since 2009, which is
caused by a mutation in KCNJ 10 and is associated, among other things, with epilepsy,
ataxia, hearing loss, metabolic alkalosis and renal salt loss.
A new disease has now been discovered that is due to a mutation in KCNJ 16 and causes
symptoms similar to the already known EAST/SeSAME syndrome. To date, six affected
patients have been identified with five different mutations (missense and nonsense
mutations) in KCNJ 16, whose symptoms and time of diagnosis are different. Two of the
patients have a homozygous mutation in KCNJ 16 (T64I or R137C), the other four
patients are compound heterozygous (in two patients I132R, R176X; in two patients
R137C, R176X; in one patient I132R, P250L). The mutations lead to changed amino
acids at the C and N terminus as well as in the selectivity filter of the potassium channel.
Patients suffer from hypokalemia, sensorineural hearing loss, renal salt wasting and,
unlike patients with EAST/SeSAME syndrome, metabolic acidosis.
In order to understand the pathomechanism behind it in more detail, the present work
examined the heteromeric channel Kir 4.2/5.1 (KCNJ 15/16), which is mainly expressed
in the proximal tubule of the kidney, where the pH value is also regulated.
To carry out the experiments, KCNJ 16 was genetically modified using side directed
mutagenesis so that the bases of the DNA were exchanged according to the identified
mutations.
Protein interaction studies using proximity ligation assay and co-immunoprecipitation
were performed to investigate the interaction between the channel subunits Kir 4.2 and
Kir 5.1. Human embryonic kidney cells (HEK) and immortalized kidney fibroblasts (Cos7)
were transfected with the genetically modified DNA and incubated in cell culture so that
they synthesized and expressed the mutated channels. This made it possible to
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demonstrate that Kir 4.2 interacts with both the wild type and all five mutated forms of
Kir 5.1 and that the patients' symptoms cannot be explained by a complete lack of
interaction between the channel subunits.
Luminescence experiments were then carried out with Xenopus laevis oocytes to
compare the quantity of expressed channels of the mutated variants with that of the wildtype
channels. The genetically modified RNA was injected into the oocytes so that they
expressed the corresponding channels with the identified mutations. These experiments
showed that quantitatively fewer of the mutated channels reached the cell surface than
the wild-type channels. The lower channel density is one of the reasons for a lower ion
current of potassium through the cell membrane and can therefore explain the symptoms
this causes in those affected.
In the present work, research was carried out on the first known disease that can be
traced back to a mutation in KCNJ 16. It thus provides important new information for a
deeper understanding of Kir 5.1, the function of inward rectifiers in the kidney and the
associated electrolyte and acid-base balance of the body.
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Dates
Created: 2024Issued: 2024-10-30Updated: 2024-10-30
Faculty
Medizin
Publisher
Philipps-Universität Marburg
Language
ger
Data types
DoctoralThesis
DFG-subjects
Kir 5.1Kir 4.2KCNJ 16KCNJ 15
DDC-Numbers
610
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Schilling, Franziska: Die Bedeutung von KCNJ 15 / KCNJ 16 -Interaktionen bei einer neuartigen komplexen tubulären Nierenerkrankung. : Philipps-Universität Marburg 2024-10-30. DOI: https://doi.org/10.17192/z2024.0340.
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This item has been published with the following license: In Copyright