Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: A. 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. 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). C. 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). D. 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 .

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Positive Control, Negative Control, Western Blot, Labeling, Marker, Electron Microscopy, Cell Culture, Immunolabeling, Transfection, Binding Assay, Staining

Journal: PLoS ONE

Article Title: Putative Structural and Functional Coupling of the Mitochondrial BK Ca Channel to the Respiratory Chain

doi: 10.1371/journal.pone.0068125

Figure Lengend Snippet: Two-dimensional separation was performed as described in the , 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; Sc 1 and Sc 2 , complexes with higher molecular weights containing cytochrome c oxidase. A typical immunoblot from three separate experiments is shown.

Article Snippet: The immunoreactions consisted of sequential incubations with a rabbit polyclonal anti-BK Ca channel β subunit 4 antibody (anti-β4, 1∶20, Alomone Labs) followed by species-specific donkey secondary antibodies coupled to 10 nm gold particles (Electron Microscopy Sciences).

Techniques: Staining, Isolation, Blue Native PAGE, Western Blot

Journal: The Journal of physiology

Article Title: MitoBK Ca channel is functionally associated with its regulatory β1 subunit in cardiac mitochondria

doi: 10.1113/JP277769

Figure Lengend Snippet: A) Immunoprecipitation (IP) of BKCa alpha and BK-β1 subunit with an antibody against BKCa alpha (see methods). Western blots revealed the presence of GRIM 19 and SERCA as markers of mitochondrial and sarcoplasmic reticulum membranes (4A, lower panels). *The signal of BK-β1 in the whole lysate was detected with chemiluminescent (ECL) substrate Supersignal™ West Femto Maximun sensitivity (see methods). Taken together, these results indicates an association between mitoBKCa and its auxiliary β1 subunit. Having demonstrated this, we characterized the biophysical properties of mitoBKCa in Wt and β1-KO cardiac mitoplasts. B) Single-channel currents of mitoBKCa recorded from Wt mitoplasts. The Po of the channel was 0.9 ± 0.08, n=4, in average at +80 mV and the indicated matrix Ca2+. C) Representative traces of a mitoplast from the β1 KO, were no single-channel current was detected at the indicated voltages and matrix Ca2+ (upper traces). Few mitoplasts (5 out of 28, mitoplasts, n=5 hearts) showed mitoBKCa channel activity with a Po of 0.4 ± 0.004, n=3 at +40 mV in the indicated matrix Ca2+. D) Plots of I vs. V at 12 µM matrix Ca2+ from Wt and β1 KO mitoplasts (data from 6 and 3 patches, respectively). A slope conductance of 302±26 (n=4) and 335 ±15 pS (n=3) was calculated for Wt and β1 KO, respectively. E) Plots of Po vs. V for mitoBKCa channels, data points represent the average of 4 and 3 mitoplasts for Wt and BK-β1 KO, respectively. Bars SEM.

Article Snippet: Polyclonal antibody against BK Ca (Rabbit Anti-KCNMA1 Cat. APC-021 and APC-151), and polyclonal antibody for BK-β1 subunit (Rabbit Anti-KCNMB1, Cat. APC-036) were from Alomone labs.

Techniques: Immunoprecipitation, Western Blot, Activity Assay

Journal: Mitochondrion

Article Title: The mitochondrial BK Ca channel cardiac interactome reveals BK Ca association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator

doi: 10.1016/j.mito.2016.08.017

Figure Lengend Snippet: BK Ca channel-interacting mitochondrial proteins identified by LC/MS/MS

Article Snippet: Antibodies The following antibodies were used: Anti-BK Ca monoclonal antibody (mAb) (75-022, UC Davis/ NIH NeuroMab facility), Anti-BK Ca polyclonal (p) Ab (APC-021, Alomone Labs), Anti-c-Myc mAb (M4439, Sigma), Anti-c-Myc polyclonal pAb (C3956, Sigma), Anti-HA mAb (H3663, Sigma), Anti-HA pAb (H6908, Sigma), anti-DDK mAb (TA50011, OriGene), Goat Anti-Rabbit IgG Alexa Fluor® 680 conjugate (A21109, Invitrogen), and IRDye® 800CW Goat Anti-Mouse IgG (926-32210, Odyssey).

Techniques: Activity Assay

Journal: Mitochondrion

Article Title: The mitochondrial BK Ca channel cardiac interactome reveals BK Ca association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator

doi: 10.1016/j.mito.2016.08.017

Figure Lengend Snippet: BK Ca channel-interacting proteins from identified by LC/MS/MS.

Article Snippet: Antibodies The following antibodies were used: Anti-BK Ca monoclonal antibody (mAb) (75-022, UC Davis/ NIH NeuroMab facility), Anti-BK Ca polyclonal (p) Ab (APC-021, Alomone Labs), Anti-c-Myc mAb (M4439, Sigma), Anti-c-Myc polyclonal pAb (C3956, Sigma), Anti-HA mAb (H3663, Sigma), Anti-HA pAb (H6908, Sigma), anti-DDK mAb (TA50011, OriGene), Goat Anti-Rabbit IgG Alexa Fluor® 680 conjugate (A21109, Invitrogen), and IRDye® 800CW Goat Anti-Mouse IgG (926-32210, Odyssey).

Techniques: Coagulation, Histone Deacetylase Assay, Sequencing