Single-Channel Properties of the ROMK-Pore-Forming Subunit of the Mitochondrial ATP-Sensitive Potassium Channel.

An increased flux of potassium ions into the mitochondrial matrix through the ATP-sensitive potassium channel (mitoKATP) has been shown to provide protection against ischemia-reperfusion injury. Recently, it was proposed that the mitochondrial-targeted isoform of the renal outer medullary potassium channel (ROMK) protein creates a pore-forming subunit of mitoKATP in heart mitochondria. Our research focuses on the properties of mitoKATP from heart-derived H9c2 cells. For the first time, we detected single-channel activity and describe the pharmacology of mitoKATP in the H9c2 heart-derived cells. The patch-clamping of mitoplasts from wild type (WT) and cells overexpressing ROMK2 revealed the existence of a potassium channel that exhibits the same basic properties previously attributed to mitoKATP. ROMK2 overexpression resulted in a significant increase of mitoKATP activity. The conductance of both channels in symmetric 150/150 mM KCl was around 97 ± 2 pS in WT cells and 94 ± 3 pS in cells overexpressing ROMK2. The channels were inhibited by 5-hydroxydecanoic acid (a mitoKATP inhibitor) and by Tertiapin Q (an inhibitor of both the ROMK-type channels and mitoKATP). Additionally, mitoKATP from cells overexpressing ROMK2 were inhibited by ATP/Mg2+ and activated by diazoxide. We used an assay based on proteinase K to examine the topology of the channel in the inner mitochondrial membrane and found that both termini of the protein localized to the mitochondrial matrix. We conclude that the observed activity of the channel formed by the ROMK protein corresponds to the electrophysiological and pharmacological properties of mitoKATP.

Evidence for a mitochondrial ATP-regulated potassium channel in human dermal fibroblasts.

Mitochondrial ATP-regulated potassium channels are present in the inner membrane of the mitochondria of various cells. In the present study, we show for the first time mitochondrial ATP-regulated potassium channels in human dermal fibroblast cells. Using the patch-clamp technique on the inner mitochondrial membrane of fibroblasts, we detected a potassium channel with a mean conductance equal to 100 pS in symmetric 150 mM KCl. The activity of this channel was inhibited by a complex of ATP/Mg2+ and activated by potassium channel openers such as diazoxide or BMS 191095. Channel activity was inhibited by antidiabetic sulfonylurea glibenclamide and 5-hydroxydecanoic acid. The influence of substances modulating ATP-regulated potassium channel activity on oxygen consumption and membrane potential of isolated fibroblast mitochondria was also studied. Additionally, the potassium channel opener diazoxide lowered the amount of superoxide formed in isolated fibroblast mitochondria. Using reverse transcriptase-PCR, we found an mRNA transcript for the KCNJ1(ROMK) channel. The presence of ROMK protein was observed in the inner mitochondrial membrane fraction. Moreover, colocalization of the ROMK protein and a mitochondrial marker in the mitochondria of fibroblast cells was shown by immunofluorescence. In summary, the ATP-regulated mitochondrial potassium channel in a dermal fibroblast cell line have been identified.

Mitochondrial potassium channels - an overview.

Mitochondria play a fundamental role in ATP synthesis within the majority of mammalian cells. Potassium channels present in the inner mitochondrial membrane are fine regulators of mitochondrial function, based on inner membrane K+ permeability. These channels are regulated by a plethora of factors and conditions in a way similar to plasma membrane potassium channels. Regulators of mitochondrial potassium channels include the membrane potential, calcium ions, free fatty acids and ATP levels within the cells. Recently, it was shown that these channels are regulated by the respiratory chain, stretching of the membrane and phosphorylation. The essential interest that has driven studies of mitochondrial potassium channels for nearly 25 years is their role in cytoprotection and in cell death. Mitochondrial potassium channels have been described in neurons, astrocytoma, cardiac and skeletal muscles, fibroblasts, keratinocytes and endothelial cells. In this overview, we summarize the current knowledge of mitochondrial potassium channels. This summary will be done with a special focus on studies performed over the last 20 years in the Laboratory of Intracellular Ion Channels at the Nencki Institute. These include studies on the electrophysiological and pharmacological properties of mitochondrial potassium channels and on their regulation by endogenous intracellular substances. Additionally, the regulation of mitochondrial potassium channels by the respiratory chain and by stretching of the inner mitochondrial membrane will be reviewed. Properties of mitochondrial potassium channels in various organisms will also be summarized.

What do we not know about mitochondrial potassium channels?

In this review, we summarize our knowledge about mitochondrial potassium channels, with a special focus on unanswered questions in this field. The following potassium channels have been well described in the inner mitochondrial membrane: ATP-regulated potassium channel, Ca(2+)-activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the two-pore domain TASK-3 potassium channel. The primary functional roles of these channels include regulation of mitochondrial respiration and the alteration of membrane potential. Additionally, they modulate the mitochondrial matrix volume and the synthesis of reactive oxygen species by mitochondria. Mitochondrial potassium channels are believed to contribute to cytoprotection and cell death. In this paper, we discuss fundamental issues concerning mitochondrial potassium channels: their molecular identity, channel pharmacology and functional properties. Attention will be given to the current problems present in our understanding of the nature of mitochondrial potassium channels. This article is part of a Special Issue entitled 'EBEC 2016: 19th European Bioenergetics Conference, Riva del Garda, Italy, July 2-6, 2016', edited by Prof. Paolo Bernardi.

[Topology of the mitochondrial potassium ion channels]. / Topologia mitochondrialnych kanalów potasowych.

In the inner mitochondrial membrane several potassium channels have been identified whose activation lead to cytoprotection during ischemic event. It was found that activation of mitochondrial large conductance calcium activated potassium channel (mitoBKCa) and ATP regulated potassium channel (mitoKATP) preserves brain and heart muscle cells against ischemia/reperfusion induced damage. However the detailed cytoprotection mechanism remains unclear. Similarly, the molecular structures and protein interactions of the mitochondrial potassium channels are still unknown. In this article, we summarize the current knowledge of the mitoKATP and mitoBKCa channels topology. Different aspects of this topic are discussed like import and assembly of the channel subunits and biophysical properties of mitochondrial compartments. Additionally, the consequences of different topology models on the cytoprotective function of the mitochondrial potassium channels were analyzed.

Quality Management in Polish Biobanking Network-Current Status Before the Implementation of Unified and Harmonized Integrated Quality Management System.

In 2017, Polish Biobanking Network was established in Poland, within BBMRI.pl project titled "Organization of Polish Biobanking Network within the Biobanking and Biomolecular Resources Research Infrastructure BBMRI-ERIC" as a strategic scientific infrastructure concept. One of the key elements of the project was the verification of the current status of QMS in the Polish biobanking institutions and the implementation of common solutions. The main goal was to indicate the current QMS level and determine the starting points for QMS development for each biobank of the Polish Biobanking Network (PBN). Within 3 years, 35 audit visits were performed. The current status and the level of QMS implementation in each biobank were assessed. Five hundred and seventy recommendations were prepared. The data was analyzed using Fischer Exact test to determine whether or not a significant association was observed. Three areas of analysis were covered: (1) BBMRI.pl status, (2) QMS implementation level and (3) private/public party, respectively. The results were discussed within 15 areas. Concluding remarks showed that some differences were observed in the case of subgroups analysis. There is convergence in QMS within the biobanks where Tissue Banks are located. Moreover, some discrepancies between the QMS implementation level in BBMRI.pl Consortium biobanks and PBN biobanks are observed. Nevertheless, the consortium members are obliged to prepare other biobanks willing to enter the PBN as Members/Observers or which already are in the PBN, so that they can meet the requirements of the quality management system that will enable efficient management of biobanking processes in these units. That is why some actions within BBMRI.pl projects are organized to help the whole biobanking community in Poland implement the harmonized solution.

Mitochondrial large-conductance potassium channel from Dictyostelium discoideum.

In the present study, we describe the existence of a large-conductance calcium-activated potassium (BKCa) channel in the mitochondria of Dictyostelium discoideum. A single-channel current was recorded in a reconstituted system, using planar lipid bilayers. The large-conductance potassium channel activity of 258±12 pS was recorded in a 50/150 mM KCl gradient solution. The probability of channel opening (the channel activity) was increased by calcium ions and NS1619 (potassium channel opener) and reduced by iberiotoxin (BKCa channel inhibitor). The substances known to modulate BKCa channel activity influenced the bioenergetics of D. discoideum mitochondria. In isolated mitochondria, NS1619 and NS11021 stimulated non-phosphorylating respiration and depolarized membrane potential, indicating the channel activation. These effects were blocked by iberiotoxin and paxilline. Moreover, the activation of the channel resulted in attenuation of superoxide formation, but its inhibition had the opposite effect. Immunological analysis with antibodies raised against mammalian BKCa channel subunits detected a pore-forming α subunit and auxiliary ß subunits of the channel in D. discoideum mitochondria. In conclusion, we show for the first time that mitochondria of D. discoideum, a unicellular ameboid protozoon that facultatively forms multicellular structures, contain a large-conductance calcium-activated potassium channel with electrophysiological, biochemical and molecular properties similar to those of the channels previously described in mammalian and plant mitochondria.

Potassium channel in the mitochondria of human keratinocytes.

The activation of mitochondrial potassium channels induces cytoprotection in various cell types. Hence, the identification of ion channels present in the inner mitochondrial membrane of keratinocytes is important in distinguishing possible protective mechanisms in these cells. In this paper, inner membrane mitochondrial ion channels of the human keratinocyte HaCaT cell line were investigated using a patch-clamp technique. We observed potassium-selective channel activity with a conductance of 83 pS at positive voltages. The I-V curve indicates that the observed channel has rectifying properties. Moreover, the channel activity was inhibited by acidic pH and 1 mM lidocaine. Using reverse transcriptase-PCR, we found an mRNA transcript for the TASK-3 (tandem pore domain acid-sensitive K channels) channel. We observed co-localization of the TASK-3 protein and a mitochondrial marker in the mitochondria of HaCaT cells. Additionally, we showed that TASK-3 knockdown HaCaT cells markedly decreased viability after UVB radiation exposure compared with control cells. In summary, the single-channel activity and properties of a mitochondrial potassium channel in a keratinocyte HaCaT cell line have been described.