Mitochondrialny kanał potasowy typu BKCa o dużym przewodnictwie aktywowany jonami wapnia – budowa i interakcje : praca doktorska

Gałecka, Shur Karolina

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Mitochondrialny kanał potasowy typu BKCa o dużym przewodnictwie aktywowany jonami wapnia – budowa i interakcje : praca doktorska

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Title: Mitochondrialny kanał potasowy typu BKCa o dużym przewodnictwie aktywowany jonami wapnia – budowa i interakcje : praca doktorska

Abstract:

Mitochondrial potassium channels, located in the inner mitochondrial membrane, play an important role in the regulation of mitochondrial function. These channels, present in various tissues, such as the heart, brain and skeletal muscles, control the influx of potassium ions into the mitochondrial matrix, which affects the volume of mitochondria, the depolarization of inner mitochondrial membrane, the rate of oxygen consumption and the synthesis of reactive oxygen species. Activation of mitochondrial potassium channels protects cells against ischemia/reperfusion damage and regulates the metabolism of cancer cells. The main research object in this thesis was the mitochondrial large conductance calcium‑activated potassium channel (mitoBKCa). Activation of this channel increases cell survival during ischemia/reperfusion of heart or brain cells. The basic biophysical and pharmacological properties of the mitoBKCa channel correspond to those of plasma membrane BKCa channels. This channel is composed of α subunits and regulatory β-type subunits. In the first part of the thesis, the hypothesis that the VEDEC isoform of the α subunit forms the mitoBKCa channel was verified. This isoform is directed to mitochondria. It is a product of the KCNMA1 gene alternative splicing – the same gene that encodes the BKCa channels from plasma membrane. To this day there was no direct evidence that this protein can form a functional channel in mitochondria. In this study, HEK293T cells were used to express the VEDEC variant. Than the channel activity in mitochondria was recorded using mitoplast patch-clamp technique., It was found for the first time that transient expression of the VEDEC isoform leads to channel activity with a conductance of 290 ± 3 pS. The channel was voltage-dependent and activated by calcium ions. Channel activity was activated by NS11021 and inhibited by hemin and paxilline, known modulators of BKCa channels. Immunofluorescence staining confirmed partial colocalization of the channel with mitochondria. The obtained data confirmed that the VEDEC isoform of the BKCa channel forms a functional channel in the inner mitochondrial membrane. Additionally, the experiments showed that HEK293T cells are a promising experimental model for the study of mitochondrial potassium channels. In the second part of the thesis, an attempt was made to identify new protein partners of the β4 regulatory subunit of the BKCa channel. Protein partners were biotinylated using TurboID ligase fused to the β4 regulatory subunit of the BKCa channel. Identification of biotinylated proteins using mass spectrometry revealed potential partners of BKCa channels. One of the proteins interacting with the BKCa channel was the TMX1. The protein is located in the ER-mitochondria contact sites. Coimmunoprecipitation and Western Blot analysis of the protein showed an interaction of this protein not only with the β4 subunit, but also with the VEDEC isoform of the α subunit.