Calcium Ions Regulate K+ Uptake into Brain Mitochondria: The Evidence for a Novel Potassium Channel

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca2+ additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca2+ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BKCa channel). Furthermore, NS1619 – a BKCa channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca2+. These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-β4 subunit (of the BKCa channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β4 subunit in neuronal mitochondria. We hypothesize that the mitochondrial BKCa channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival

Alternative Targets for Modulators of Mitochondrial Potassium Channels

Mitochondrial potassium channels control potassium influx into the mitochondrial matrix and thus regulate mitochondrial membrane potential, volume, respiration, and synthesis of reactive oxygen species (ROS). It has been found that pharmacological activation of mitochondrial potassium channels during ischemia/reperfusion (I/R) injury activates cytoprotective mechanisms resulting in increased cell survival. In cancer cells, the inhibition of these channels leads to increased cell death. Therefore, mitochondrial potassium channels are intriguing targets for the development of new pharmacological strategies. In most cases, however, the substances that modulate the mitochondrial potassium channels have a few alternative targets in the cell. This may result in unexpected or unwanted effects induced by these compounds. In our review, we briefly present the various classes of mitochondrial potassium (mitoK) channels and describe the chemical compounds that modulate their activity. We also describe examples of the multidirectional activity of the activators and inhibitors of mitochondrial potassium channels

The mitochondrial protein import machinery has multiple connections to the respiratory chain

AbstractThe mitochondrial inner membrane harbors the complexes of the respiratory chain and protein translocases required for the import of mitochondrial precursor proteins. These complexes are functionally interdependent, as the import of respiratory chain precursor proteins across and into the inner membrane requires the membrane potential. Vice versa the membrane potential is generated by the proton pumping complexes of the respiratory chain. Besides this basic codependency four different systems for protein import, processing and assembly show further connections to the respiratory chain. The mitochondrial intermembrane space import and assembly machinery oxidizes cysteine residues within the imported precursor proteins and is able to donate the liberated electrons to the respiratory chain. The presequence translocase of the inner membrane physically interacts with the respiratory chain. The mitochondrial processing peptidase is homologous to respiratory chain subunits and the carrier translocase of the inner membrane even shares a subunit with the respiratory chain. In this review we will summarize the import of mitochondrial precursor proteins and highlight these special links between the mitochondrial protein import machinery and the respiratory chain. This article is part of a Special Issue entitled: Respiratory complex II: Role in cellular physiology and disease

Mitochondrial BK Channel Openers CGS7181 and CGS7184 Exhibit Cytotoxic Properties

Potassium channel openers (KCOs) have been shown to play a role in cytoprotection through the activation of mitochondrial potassium channels. Recently, in several reports, a number of data has been described as off-target actions for KCOs. In the present study, we investigated the effects of BKCa channel openers CGS7181, CGS7184, NS1619, and NS004 in neuronal cells. For the purpose of this research, we used a rat brain, the mouse hippocampal HT22 cells, and the human astrocytoma U-87 MG cell line. We showed that CGS7184 activated the mitochondrial BKCa (mitoBKCa) channel in single-channel recordings performed on astrocytoma mitoplasts. Moreover, when applied to the rat brain homogenate or isolated rat brain mitochondria, CGS7184 increased the oxygen consumption rate, and can thus be considered a potentially cytoprotective agent. However, experiments on intact neuronal HT22 cells revealed that both CGS7181 and CGS7184 induced HT22 cell death in a concentration- and time-dependent manner. By contrast, we did not observe cell death when NS1619 or NS004 was applied. CGS7184 toxicity was not abolished by BKCa channel inhibitors, suggesting that the observed effects were independent of a BKCa-type channel activity. CGS7184 treatment resulted in an increase of cytoplasmic Ca2+ concentration that likely involved efflux from internal calcium stores and the activation of calpains (calcium-dependent proteases). The cytotoxic effect of the channel opener was partially reversed by a calpain inhibitor. Our data show that KCOs under study not only activate mitoBKCa channels from brain tissue, but also induce cell death when used in cellular models

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