Oddziaływania kanału potasowego ROMK2 z białkami mitochondrialnymi : praca doktorska

Krajewska, Milena.

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Oddziaływania kanału potasowego ROMK2 z białkami mitochondrialnymi : praca doktorska

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Title: Oddziaływania kanału potasowego ROMK2 z białkami mitochondrialnymi : praca doktorska

Abstract:

Hypoxia/reperfusion of the heart and brain tissues leads to cell injury and necrosis and is the most common cause of death in developed countries. Mitochondrial channels are implicated in cytoprotection during reperfusion. One of them is the mitochondrial potassium channel inhibited by ATP (mitoKATP). It was proposed that the ROMK2 protein may be a pore-forming subunit of the mitoKATP channel. ROMK2 is an isoform of the ROMK1 protein that forms rectifying potassium channels in the plasma membrane. These channels interact with many proteins involved in the regulation of their activity, or in their trafficking. However, protein partners of the ROMK2 channel have not been identified so far. The main aim of the research presented in this doctoral dissertation was the identification of proteins that are part of the mitochondrial proxisome of the ROMK2 channel. For this purpose, proximity biotinylation was used. Among the biotinylated proteins, cytoplasmic proteins have been identified that participate among others in endocytosis, vesicular transport, oxidative stress, nucleotide synthesis, or lipid metabolism. In addition, subunits of inner and outer mitochondrial membrane translocases have been identified. These complexes allow the import of nuclear-encoded proteins into mitochondria. Among them, acylglycerol kinase (AGK) was found, which is involved in the transport of polytopic proteins into the inner mitochondrial membrane. AGK is a dual-function protein and participates also in the synthesis of lipids: lysophosphatidic acid (LPA) and phosphatidic acid (PA). The occurrence of a complex containing ROMK2 and AGK was supported by co-immunoprecipitation. Another goal was to investigate the pharmacological modulation of ROMK2 channel activity. To carry out these studies the ROMK2-6xHis protein was produced in bacteria Escherichia coli; bacterial membranes were solubilized with amphipathic copolymers, and the channel protein was purified by immobilized metal affinity chromatography. To study the electrical activity of ROMK2-6xHis the planar lipid bilayer technique was used. The impact of known mitoKATP channel modulators on the activity of the ROMK2 protein was tested. The ROMK2 channel was activated by the mitoKATP activator – diazoxide and blocked by mitoKATP inhibitors, i.e. ATP/Mg2+, 5-HD, and glibenclamide. These results confirmed that the ROMK2 protein may be a pore-forming subunit of mitoKATP. Additionally, it was shown that the influence of these modulators was not related to the presence of accessory proteins of ROMK channels. Finally, a functional interaction between ROMK2 and AGK was demonstrated. The activity of the ROMK2 channel was regulated by the products of the enzymatic activity of the AGK protein, i.e. LPA and PA.