Tumor cells are resistant to hypoxia but the underlying mechanism(s) of this tolerance remain poorly understood. In healthy brain cells, plasmalemmal Ca(2+)-activated K(+) channels ((plasma)BK) function as oxygen sensors and close under hypoxic conditions. Similarly, BK channels in the mitochondrial inner membrane ((mito)BK) are also hypoxia sensitive and regulate reactive oxygen species production and also permeability transition pore formation. Both channel populations are therefore well situated to mediate cellular responses to hypoxia. In tumors, BK channel expression increases with malignancy, suggesting these channels contribute to tumor growth; therefore, we hypothesized that the sensitivity of (plasma)BK and/or (mito)BK to hypoxia differs between glioma and healthy brain cells. To test this, we examined the electrophysiological properties of (plasma)BK and (mito)BK from a human glioma cell line during normoxia and hypoxia. We observed single channel activities in whole cells and isolated mitoplasts with slope conductance of 199?±?8 and 278?±?10 pA, respectively. These currents were Ca(2+)- and voltage-dependent, and were inhibited by the BK channel antagonist charybdotoxin (0.1 ?M). (plasma)BK could only be activated at membrane potentials >+40 mV and had a low open probability (NPo) that was unchanged by hypoxia. Conversely, (mito)BK were active across a range of membrane potentials (-40 to +40 mV) and their NPo increased during hypoxia. Activating (plasma)BK, but not (mito)BK induced cell death and this effect was enhanced during hypoxia. We conclude that unlike in healthy brain cells, glioma (mito)BK channels, but not (plasma)BK channels are oxygen sensitive.
Polyunsaturated fatty acids (PUFAs) and their metabolites can modulate several biochemical processes in the cell and thus prevent various diseases. PUFAs have a number of cellular targets, including membrane proteins. They can interact with plasma membrane and intracellular potassium channels. The goal of this work was to verify the interaction between PUFAs and the most common and intensively studied mitochondrial large conductance Ca(2+)-regulated potassium channel (mitoBKCa). For this purpose human astrocytoma U87 MG cell lines were investigated using a patch-clamp technique. We analyzed the effects of arachidonic acid (AA); eicosatetraynoic acid (ETYA), which is a non-metabolizable analog of AA; docosahexaenoic acid (DHA); and eicosapentaenoic acid (EPA). The open probability (Po) of the channel did not change significantly after application of 10?M ETYA. Po increased, however, after adding 10?M AA. The application of 30?M DHA or 10?M EPA also increased the Po of the channel. Additionally, the number of open channels in the patch increased in the presence of 30?M EPA. Collectively, our results indicate that PUFAs regulate the BKCa channel from the inner mitochondrial membrane.
Several studies have proposed that the antibiotic minocycline (MC) has cytoprotective activities. Nevertheless, when cells have been exposed to MC at micromolar concentrations, detrimental effects have been also observed. Despite the known inhibitory activity of MC on ATP synthesis and the Ca(2+) retention capacity of isolated rat liver and brain mitochondria, the underlying mechanism is still debated. Here, we present further arguments supporting our concept that MC acting on rat liver mitochondria suspended in KCl medium permeabilizes the inner membrane. Supplementation of the medium with cytochrome c and NAD(+) strongly enhanced the respiration of MC-treated mitochondria, thus partly preventing or reversing the inhibitory effect of MC on state 3 or uncoupled respiration. These results indicate that MC produced depletion of mitochondrial cytochrome c and NAD(+) , thus impairing mitochondrial respiration. In addition, NADH oxidation by alamethicin-permeabilized mitochondria supplemented with cytochrome c was insensitive to 200 ?m MC, arguing against direct impairment of respiratory chain complexes by MC. Finally, a surprising feature of MC was its accumulation or binding by intact rat liver mitochondria, but not by mitochondria permeabilized with alamethicin or disrupted by freezing and thawing.
Since about 60 years a phenomenon now called permeability transition is known in mitochondria. It involves a large pore in the inner mitochondrial membrane, the permeability transition pore (PTP) whose molecular structure is still unknown. Year after year, new hypotheses have been developed how this pore could look like and which proteins cold be structural elements. Enormous progress was made in understanding function, rich pharmacology, and possible biochemical modulation of the PTP. However, many of the structural hypotheses that seemed to be well established by experiments had to be rejected later after their falsification by further experiments. The aim of this review is to give a brief insight into confirmed and less known details of the nature of the pore and of its function. Thereafter, this review will critically report about some of the unknown elements and hypotheses that had to be rejected.
Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+)-regulated potassium channel (mitoBKCa channel) was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma) U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+) at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel ?4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the ?4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.
Mitochondria are crucially involved in the intrinsic pathway of apoptosis. Upon induction of apoptosis, proapoptotic proteins of the Bcl-2 family, in particular Bax and Bak, transfer the death signal to the organelle. The outcome is release of proapoptotic factors, such as cytochrome c, and mitochondrial changes, such as depolarization. Details of the mechanism by which Bax mediates mitochondrial alterations, however, are unknown. Using the single-channel patch-clamp method, we studied mitoplasts (vesicles of inner membrane) from rat astrocyte and liver mitochondria and intact murine glioma mitochondria to determine the action of proapoptotic Bax and antiapoptotic Bcl-xL on the mitochondrial Ca(2+)-activated channel (mtBK) and the permeability transition pore (mtPTP). Bax (1 nM) inhibited the open probability of the mtBK, whereas Bcl-xL or control proteins had no effect. Incubating mitochondria with iberiotoxin, an inhibitor of mtBK, induced the release of cytochrome c. Bcl-xL inhibited the effects of Bax on mtBK. Furthermore, in patch-clamp studies Bcl-xL inhibited the mtPTP itself, whereas Bax had no direct effect on the mtPTP. We conclude that Bax exerts its proapototic effect by inhibiting mitochondrial K(+) channels, whereas Bcl-xL exerts its antiapoptotic effect by inhibiting the effects of Bax on mitochondrial potassium channels and by direct inhibition of the mtPTP.
Ropinirole, an agonist of the post-synaptic dopamine D2-receptor, exerts neuroprotective activity. The mechanism is still under discussion. Assuming that this neuroprotection might be associated with inhibition of the apoptotic cascade underlying cell death, we examined a possible effect of ropinirole on the permeability transition pore (mtPTP) in the mitochondrial inner membrane. Using isolated rat liver mitochondria, the effect of ropinirole was studied on Ca2+-triggered large amplitude swelling, membrane depolarization and cytochrome c release. In addition, the effect of ropinirole on oxidation of added, membrane-impermeable NADH was investigated. The results revealed doubtlessly, that ropinirole can inhibit permeability transition. In patch-clamp experiments on mitoplasts, we show directly that ropinirole interacts with the mtPTP. Thus, ropinirole reversibly inhibits the opening of mtPTP with an IC50 of 3.4 microM and a Hill coefficient of 1.3. In both systems (i.e. energized mitochondria and mitoplasts) the inhibitory effect on permeability transition was attenuated by increasing concentrations of inorganic phosphate. In addition, we showed with antimycin A-treated mitochondria that ropinirole failed to suppress respiratory chain-linked reactive oxygen species release. In conclusion, our data suggest that the neuroprotective activity of ropinirole is due to the blockade of the Ca2+-triggered permeability transition.
Three types of potassium channels cooperate with the permeability transition pore (PTP) in the inner mitochondrial membranes of various tissues, mtK((ATP)), mtBK, and mtKv1.3. While the latter two share similarities with their plasma membrane counterparts, mtK((ATP)) exhibits considerable differences with the plasma membrane K((ATP))-channel. One important function seems to be suppression of release of proapototic substances from mitochondria through the PTP. Open potassium channels tend to keep the PTP closed thus acting as antiapoptotic. Nevertheless, in their mode of action there are considerable differences among them. This review introduces three K(+)-channels and the PTP, and discusses known facts about their interaction.
The semi-synthetic tetracycline derivative minocycline exerts neuroprotective properties in various animal models of neurodegenerative disorders. Although anti-inflammatory and anti-apoptotic effects are reported to contribute to the neuroprotective action, the exact molecular mechanisms underlying the beneficial properties of minocycline remain to be clarified. We analyzed the effects of minocycline in a cell culture model of neuronal damage and in single-channel measurements on isolated mitoplasts. Treatment of neuron-enriched cortical cultures with rotenone, a high affinity inhibitor of the mitochondrial complex I, resulted in a deregulation of the intracellular Ca2+-dynamics, as recorded by live cell imaging. Minocycline (100 microM) and cyclosporin A (2 microM), a known inhibitor of the mitochondrial permeability transition pore, decreased the rotenone-induced Ca2+-deregulation by 60.9% and 37.6%, respectively. Investigations of the mitochondrial permeability transition pore by patch-clamp techniques revealed for the first time a dose-dependent reduction of the open probability by minocycline (IC(50)=190 nM). Additionally, we provide evidence for the high antioxidant potential of MC in our model. In conclusion, the present data substantiate the beneficial properties of minocycline as promising neuroprotectant by its inhibitory activity on the mitochondrial permeability transition pore.
There is an ongoing debate on the presence of beneficial effects of minocycline (MC), a tetracycline-like antibiotic, on the preservation of mitochondrial functions under conditions promoting mitochondria-mediated apoptosis. Here, we present a multiparameter study on the effects of MC on isolated rat liver mitochondria (RLM) suspended either in a KCl-based or in a sucrose-based medium. We found that the incubation medium used strongly affects the response of RLM to MC. In KCl-based medium, but not in sucrose-based medium, MC triggered mitochondrial swelling and cytochrome c release. MC-dependent swelling was associated with mitochondrial depolarization and a decrease in state 3 as well as uncoupled respiration. Swelling of RLM in KCl-based medium indicates that MC permeabilizes the inner mitochondrial membrane (IMM) to K(+) and Cl(-). This view is supported by our findings that MC-induced swelling in the KCl-based medium was partly suppressed by N,N-dicyclohexylcarbodiimide (an inhibitor of IMM-linked K(+)-transport) and tributyltin (an inhibitor of the inner membrane anion channel) and that swelling was less pronounced when RLM were suspended in choline chloride-based medium. In addition, we observed a rapid MC-induced depletion of endogenous Mg(2+) from RLM, an event that is known to activate ion-conducting pathways within the IMM. Moreover, MC abolished the Ca(2+) retention capacity of RLM irrespective of the incubation medium used, most likely by triggering permeability transition. In summary, we found that MC at low micromolar concentrations impairs several energy-dependent functions of mitochondria in vitro.
We previously demonstrated that the progesterone (PROG) metabolite allopregnanolone (AP) is more potent than PROG in the treatment of traumatic brain injury (TBI) and stroke, but the mechanisms for this differential effect are little understood. The mitochondrial permeability transition pore (mtPTP) appears to be a key player in the intrinsic pathway of apoptosis-induced loss of neurons. Its activation is accompanied by the release of cytochrome c (cyt c) from the intermembrane gap and subsequent cell death. We investigated whether mtPTP is implicated in the mechanisms of PROG and AP neuroprotection following traumatic and ischemic brain injury. To assess the neurosteroids direct effects on mtPTP activity at the single-channel level, recordings from the inner mitochondrial membrane were obtained by a patch-clamp approach in rat liver mitoplasts. AP but not PROG strongly inhibited mtPTP currents. Interaction of AP with the PTP was further supported by a swelling assay demonstrating that AP inhibited Ca(2+)-triggered swelling in functionally intact rat liver and brain mitochondria. If AP inhibits the mtPTP, it should prevent the mitochondrial cyt c release seen in stroke and TBI. To test this idea, we subjected one group of rats to cortical contusion injury (CCI) and another to transient middle cerebral artery occlusion (MCAO). AP-treated animals showed substantially decreased cyt c release and AP was more potent than PROG in inhibiting mitochondrial cyt c release at 24 h post-CCI and -MCAO. Our results demonstrate that AP inhibits the mtPTP current. This may help to explain its more potent anti-apoptotic and neuroprotective effects compared to PROG.
Existing literature on estradiol indicates that it affects mitochondrial functions at low micromolar concentrations. Particularly blockade of the permeability transition pore (PTP) or modulation of the enzymatic activity of one or more complexes of the respiratory chain were suspicious. We prepared mitoplasts from rat liver mitochondria (RLM) to study by single-channel patch-clamp techniques the PTP, and from rat astrocytes to study the potassium BK-channel said to modulate the PTP. Additionally, we measured respiration of intact RLM. After application of 17?-estradiol (?E) our single-channel results reveal a transient increase of activity of both, the BK-channel and the PTP followed by their powerful inhibition. Respiration measurements demonstrate inhibition of the Ca(2+)-induced permeability transition, as well, though only at higher concentrations (?30?M). At lower concentrations, we observed an increase of endogenous- and state 2-respiration. Furthermore, we show that ?E diminishes the phosphorylating respiration supported by complex I-substrates (glutamate/malate) or by the complex II-substrate succinate. Taken together the results suggest that ?E affects mitochondria by several modes, including partial inhibition of the activities of ion channels of the inner membrane and of respiration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).
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