SummaryMitochondrial potassium channels are believed to contribute to cytoprotection of injured cardiac and neuronal tissues. The following potassium channels have been described in the inner mitochondrial membrane: the ATP-regulated potassium channel, the large conductance Ca 21 -activated potassium channel, the voltage-gated Kv1.3 potassium channel, and the twin-pore domain TASK-3 potassium channel. The putative functional roles of these channels include changes in mitochondrial matrix volume, mitochondrial respiration, and membrane potential. In addition, the activity of these channels modulates the generation of reactive oxygen species by mitochondria. In this article, we discuss recent observations on three fundamental issues concerning mitochondrial potassium channels: (i) their molecular identity, (ii) their interaction with potassium channel openers and inhibitors, and (iii) their functional properties.

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.

Uncoupling proteins (UCPs) are mitochondrial inner membrane proteins sustaining an inducible proton conductance. They weaken the proton electrochemical gradient built up by the mitochondrial respiratory chain. Brown fat UCP1 sustains a free fatty acid (FA)-induced purine nucleotide (PN)-inhibited proton conductance. Inhibition of the proton conductance by PN has been considered as a diagnostic of UCP activity. However, conflicting results have been obtained in isolated mitochondria for UCP homologues (i.e., UCP2, UCP3, plant UCP, and protist UCP) where the FFA-activated proton conductance is poorly sensitive to PN under resting respiration conditions. Our recent work clearly indicates that the membranous coenzyme Q, through its redox state, represents a regulator of the inhibition by PN of FFA-activated UCP1 homologues under phosphorylating respiration conditions. Several physiological roles of UCPs have been suggested, including a control of the cellular energy balance as well as the preventive action against oxidative stress. In this paper, we discuss new information emerging from comparative proteomics about the impact of UCPs on mitochondrial physiology, when recombinant UCP1 is expressed in yeast and when UCP2 is over-expressed in hepatic mitochondria during steatosis.

The endothelium is considered to be relatively independent of the mitochondrial energy supply. The goals of this study were to examine mitochondrial respiratory functions in endothelial cells and isolated mitochondria and to assess the influence of chronic high glucose exposure on the aerobic metabolism of these cells. A procedure to isolate of bioenergetically active endothelial mitochondria was elaborated. Human umbilical vein endothelial cells (EA.hy926 line) were grown in medium containing either 5.5 or 25 mM glucose. The respiratory response to elevated glucose was observed in cells grown in 25 mM glucose for at least 6 days or longer. In EA.hy926 cells, growth in high glucose induced considerably lower mitochondrial respiration with glycolytic fuels, less pronounced with glutamine, and higher respiration with palmitate. The Crabtree effect was observed in both types of cells. High glucose conditions produced elevated levels of cellular Q10, increased ROS generation, increased hexokinase I, lactate dehydrogenase, acyl-CoA dehydrogenase, uncoupling protein 2 (UCP2), and superoxide dismutase 2 expression, and decreased E3-binding protein of pyruvate dehydrogenase expression. In isolated mitochondria, hyperglycaemia induced an increase in the oxidation of palmitoylcarnitine and glycerol-3-phosphate (lipid-derived fuels) and a decrease in the oxidation of pyruvate (a mitochondrial fuel); in addition, increased UCP2 activity was observed. Our results demonstrate that primarily glycolytic endothelial cells possess highly active mitochondria with a functioning energy-dissipating pathway (UCP2). High-glucose exposure induces a shift of the endothelial aerobic metabolism towards the oxidation of lipids and amino acids.Electronic supplementary materialThe online version of this article (doi:10.1007/s00424-012-1156-1) contains supplementary material, which is available to authorized users.

Bednarczyk P, Koziel A, Jarmuszkiewicz W, Szewczyk A. Large-conductance Ca 2ϩ -activated potassium channel in mitochondria of endothelial EA.hy926 cells. Am J Physiol Heart Circ Physiol 304: H1415-H1427, 2013. First published March 29, 2013 doi:10.1152/ajpheart.00976.2012In the present study, we describe the existence of a large-conductance Ca 2ϩ -activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassiumselective current was recorded with a mean conductance equal to 270 Ϯ 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 M Ca 2ϩ , 10 M NS1619, and 0.5 M NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassiumdependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a poreforming ␣-subunit and an auxiliary ␤2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calciumdependent potassium channel with properties similar to those of the surface membrane BKCa channel. mitochondria; endothelium; potassium channel; electrophysiology; bioenergetics BECAUSE ATP SUPPLIES in endothelial cells are relatively independent of mitochondrial oxidative pathways, studies on the bioenergetics of endothelial mitochondria have not been intensive to date. However, several recent observations suggest that endothelial mitochondria not only contribute to ATP generation but are also centrally involved in maintaining the fine regulatory balance among mitochondrial calcium concentrations, reactive oxygen species (ROS) production, and nitric oxide production (9, 10, 16). Endothelial mitochondria may also function as sensors of alternations in the local environment and contribute to the survival of endothelial cells under oxidative stress, and the mitochondrial ROS of endothelial cells are important signaling molecules (43).Endothelial cells are involved in many aspects of vascular ...

An uncoupling protein (UCP) has been identified in mitochondria from؉ cycling, early during phylogenesis before the major radiation of phenotypic diversity in eukaryotes and could occur in the whole eukaryotic world.

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Endothelial cells play an important physiological role in vascular homeostasis. They are also the first barrier that separates blood from deeper layers of blood vessels and extravascular tissues. Thus, they are exposed to various physiological blood components as well as challenged by pathological stimuli, which may exert harmful effects on the vascular system by stimulation of excessive generation of reactive oxygen species (ROS). The major sources of ROS are NADPH oxidase and mitochondrial respiratory chain complexes. Modulation of mitochondrial energy metabolism in endothelial cells is thought to be a promising target for therapy in various cardiovascular diseases. Uncoupling protein 2 (UCP2) is a regulator of mitochondrial ROS generation and can antagonise oxidative stress-induced endothelial dysfunction. Several studies have revealed the important role of UCP2 in hyperglycaemia-induced modifications of mitochondrial function in endothelial cells. Additionally, potassium fluxes through the inner mitochondrial membrane, which are involved in ROS synthesis, affect the mitochondrial volume and change both the mitochondrial membrane potential and the transport of calcium into the mitochondria. In this review, we concentrate on the mitochondrial role in the cytoprotection phenomena of endothelial cells.