Schematic of the proposed model and the applied substrates/inhibitors of the respiratory chain and activators/blockers of the mitoBK_Ca channel.

<p>Complexes of the respiratory chain are shown, including NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), ubiquinol cytochrome c oxidoreductase (complex III), and cytochrome c oxidase (complex IV). All of the substances (i.e., those that interact with the respiratory chain and with the mitoBK_Ca channel) that were used in this study are shown.</p

NADH reduces the P_o of the mitochondrial large-conductance Ca²⁺-regulated potassium channel at positive voltages.

<p><b>A.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 and −40 mV under control conditions, after the addition of 200 µM reduced nicotinamide adenine dinucleotide (NADH) and after perfusion. <b>B.</b> Single-channel recordings of the mitoBK_Ca channel activity in a symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 250 nM rotenone (Rot) and 200 µM NADH plus 250 nM Rot, and after perfusion. <b>C.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 1 µM antimycin A (Anti) and 200 µM NADH plus 1 µM Anti and after perfusion. <b>D.</b> Analysis of P_o under the conditions described in A, B and C. *P<0.003 vs. the control.</p

Cyanide abolishes the inhibitory effect of the respiratory chain substrates succinate and NADH.

<p><b>A.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 30 µM KCN and 5 mM succinate plus 30 µM KCN, and after perfusion. <b>B.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 mV under control conditions, after the addition of 30 µM KCN and 200 µM NADH plus 30 µM KCN, and after perfusion. <b>C.</b> Distribution of P_o at different voltages under the conditions described in A and B.</p

2D BN/SDS-PAGE separation of native astrocytoma mitochondria protein extracts.

<p>Two-dimensional separation was performed as described in the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068125#s2" target="_blank">Materials and Methods</a></i>, and the PVDF membrane was first immunoblotted for the BK_Ca channel β4 subunit (below, Coomassie staining panel). Next, the PVDF membrane was immunoblotted for the subunits of individual respiratory chain complexes (below the BK_Ca β4 panel). The BN-PAGE was calibrated based on the location of mitochondrial respiratory chain complexes that were isolated from rat heart mitochondria (above the panel for the blue native PAGE of mitochondria from astrocytoma cells). In the native astrocytoma lysate, mitochondria BK_Ca β4 co-localized with subunit I of cytochrome c oxidase. M, the monomeric form of cytochrome c oxidase; D, the dimeric form of cytochrome c oxidase; Sc₁ and Sc₂, complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.</p

Localization of the BK_Ca channel regulatory β4 subunit in astrocytoma mitochondria.

<p><b>A.</b> Detection of mitoBK_Ca channel regulatory β4 subunit mRNA in astrocytoma cells. The BK_Ca subunit β4 mRNA was detected at a size of 405 bp. No products were obtained for the BK_Ca subunits β1, β2 and β3. GAPDH served as a positive control and was detected at a size of 496 bp. The negative control without reverse transcriptase (−RT) and samples without cDNA (−A) had no signals. The results presented are representative of seven independent experiments. <b>B.</b> Immunoblot of astrocytoma mitochondria, astrocytoma cell homogenate and brain homogenate fractions labeled with the anti-BK_Ca channel β4 subunit antibody. A control antigen (BK_Ca β4+ peptide) was used as a positive control for the specificity of the antibody. An anti-cytochrome c oxidase subunit IV antibody (COX IV) was used as a mitochondrial marker (n = 3). <b>C.</b> Immuno-gold electron microscopy localization of the BK_Ca channel β4 regulatory subunit in mitochondria of cultured human astrocytoma cells. The β4 subunit molecules were labeled using 10 nm colloidal-gold particles (arrows). <b>D.</b> High-power confocal image of cultured astrocytoma cells immunolabeled to detect OxPhos (red) and β4-GFP-transfected cells (green). The superimposition of the two signals revealed the mitochondrial localization of BK_Ca β4 in human astrocytoma cells (yellow). The DNA-binding dye DAPI was used to stain the cell nuclei (blue). For details concerning the astrocytoma cells, see the <i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068125#s2" target="_blank">Materials and Methods</a></i>.</p

Effects of TMPD/ascorbate on mitoBK_Ca channel activity.

<p><b>A.</b> Single-channel recordings of the mitoBK_Ca channel activity in symmetric 150/150 mM KCl isotonic solution (200 µM Ca²⁺) at +40 and −40 mV under control conditions, after the addition of 250 µM TMPD with 500 µM Ascorbate (Asc) and after perfusion. <b>B.</b> Analysis of the probability of channel opening under the conditions described in A. *P<0.01 vs. the control.</p