Figures and data
Characterization of BKCa channels in murine and human BCCs.
(A; B) I-V curves (left) and corresponding maximal currents (right) of MMTV-PyMT WT (A) and MMTV-PyMT BK-KO cells (B), either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. n (cells) = 15 WT ctrl, 17 WT + PAX, 17 WT + IBTX, 16 BK-KO ctrl, 17 BK-KO + PAX, 19 BK-KO + IBTX. ***p≤0.001, Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s multiple comparison test. ‡p≤0.001 compared to respective WT condition, Welch’s t-test. (C) Representative fluorescence images (left) and statistics (right) of MMTV-PyMT WT and BK-KO cells loaded with the ΔΨPM sensitive dye Dibac4(3). N = 6 independent experiments, **p≤0.01, Unpaired t-test. (D) I-V curves (left) and maximal currents (right) of MDA-MB-453 cells, either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. n (cells) = 30 ctrl, 22 + PAX, 24 + IBTX. ***p≤0.001, Kruskal-Wallis test followed by Dunn’s multiple comparison test. (E) I-V curves (left) and maximal currents (right) of MCF-7 cells, either under control conditions, or in the presence of paxilline or iberiotoxin. Data shows average ± SEM. n (cells) = 16 ctrl, 20 + PAX, 15 + IBTX. (F) Schematic representation of constructs used for over-expression in MCF-7 cells. The DEC exon is indicated in green. (G) Representative images (left) of MCF-7 cells either expressing BKCaRFP (upper) or BKCa-DECRFP (lower), additionally stained with MitoGREEN. Average Pearson correlations ± SEM of MitoGREEN and RFP of BKCa or BKCa-DEC are shown. n (cells) = 17 BKCa – RFP, 22 BKCa-DECRFP. *p≤0.05, Unpaired t-test. (H) I-V curves (left and middle) and corresponding maximal currents (right) of MCF-7 cells expressing BKCa (left) or BKCa-DEC (middle), respectively, either under control conditions, or in the presence of paxilline or iberiotoxin. Data represents average ± SEM. n (cells) = 18 BKCaRFP ctrl, 14 BKCaRFP + PAX, 19 BKCaRFP + IBTX, 18 BKCa-DECRFP ctrl, 21 BKCa-DECRFP + PAX, 18 BKCa-DECRFP + IBTX. **p≤0.01, ***p≤0.001, Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s multiple comparison test. †p≤0.01 between ctrl conditions, Welch’s t-test.
BKCa modulates the subcellular Ca2+ homeostasis in BCCs.
Cytosolic (A – C), endoplasmic reticulum (ER) (D – F), and mitochondrial Ca2+ dynamics (G – I) over-time of MMTV-PyMT WT and MMTV-PyMT BK-KO cells (A, D, G), MDA-MB-453 cells (B, E, H) or MCF-7 cells (C, F, I). All data represent average ± SEM. At time points indicated in the panels, cytosolic and mitochondrial Ca2+ alterations were evoked by extracellular stimulation with ATP (A – C, G – I), or by Ca2+ depletion of the ER using Ca2+-free buffer containing the Ca2+ chelator EGTA, followed by administration of the SERCA inhibitor BHQ prior to ATP administration (D – F). MMTV-PyMT (A, D, G) and MDA-MB-453 (B, E, H) cells were either measured under control conditions, or in the presence of paxilline (+ PAX) or iberiotoxin (+ IBTX). MCF-7 cells (C, F, I) either expressed an RFP (ctrl), BKCaRFP, or BKCa-DECRFP. N (independent experiments) / n (cells analyzed) = A: 17/784 WT ctrl, 18/857 BK-KO ctrl, 6/300 WT + PAX, 6/300 BK-KO + PAX, 5/318 WT + IBTX, 5/304 BK-KO + IBTX, B: 4/151 ctrl, 4/132 + PAX, 4/87 + IBTX, C: 5/111 ctrl, 5/117 + BKCaRFP, 5/91 + BKCa-DECRFP, D: 14/116 WT ctrl, 13/117 BK-KO ctrl, 8/71 WT + PAX, 8/92 BK-KO + PAX, 6/102 WT + IBTX, 6/86 BK-KO + IBTX, E: 7/44 ctrl, 9/34 + PAX, 5/49 + IBTX, F: 4/25 ctrl, 4/35 + BKCaRFP, 4/38 + BKCa-DECRFP, G: 11/47 WT ctrl, 12/86 BK-KO ctrl, 6/46 WT + PAX, 6/58 BK-KO + PAX, 5/59 WT + IBTX, 4/43 BK-KO + IBTX, H: 8/33 ctrl, 8/28 + PAX, 5/22 + IBTX, I: 5/28 ctrl, 4/27 + BKCaRFP, 4/24 + BKCa-DECRFP. **p≤0.01, ***p≤0.001. Kruskal-Wallis test followed by Dunn’s MC test (A, B, C, D, I), One-Way ANOVA test followed by Tukey’s MC test (E, F, G) or Brown-Forsythe ANOVA test followed by Games-Howell’s MC test (H). #p≤0.05, ‡p≤0.001 compared to respective WT condition, Mann-Whitney test (A, D, ctrl in G), or Welch’s t test (+ IBTX in G).
BKCa channels alter the metabolic activity of BCCs.
(A, D) Average ECAR over-time ± SEM of MMTV-PyMT WT (A, red) and BK-KO cells (A, black), or MDA-MB-453 cells (D) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated. (B, E) Average basal and (C, F) maximal ECAR ± SEM of MMTV-PyMT WT (B, C, left) and BK-KO cells (B, C, right), or MDA-MB-453 cells (E, F) under control conditions, or in the presence of paxilline or iberiotoxin. (G, J) Average OCR over-time ± SEM of MMTV-PyMT WT (G, red) and BK-KO cells (G, black), or MDA-MB-453 cells (J) in response to administration of Oligomycin-A (+O), FCCP (+F) and Antimycin-A (+A) at time points indicated. (H, K) Average basal and (I, L) maximal OCR ± SEM of MMTV-PyMT WT (H, I, left) and BK-KO cells (H, I, right), or MDA-MB-453 cells (K, L) under control conditions, or in the presence of paxilline or iberiotoxin. (M – P) LC-MS-based determination of major glycolytic and mitochondrial metabolites in particular Lactate (M, [Lactate]), Pyruvate (N, [Pyruvate]), Asparagine (O, [Asparagine]) and Glutamine (P, [Glutamine]) of MMTV-PyMT WT (left panels), BK-KO (middle panels) and MDA-MB-453 cells (right panels), either under control conditions or after cell cultivation with paxilline. N (independent experiments) = A, B, C, G, H, I: 7 WT ctrl and BK-KO, 3 for all others, D, E, F, J, K, L: 3 for all, M – P: 7 for BK-KO ctrl, 6 for all others. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (B, E, F, I), Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s MC test (C, H), One-Way ANOVA test followed by Tukey’s MC test (K, L) or Mann-Whitney test (M – P). #p≤0.05, †p≤0.01, ‡p≤0.001, to respective WT condition, Mann-Whitney test (B, C, + IBTX in I, M – P), Welch’s t-test (ctrl in H and I) or Unpaired t-test (+ IBTX in H).
Expression of BKCa modulates mitochondrial function and glucose uptake of BCCs.
(A – D) Representative fluorescence images and -ratios (Fmito/Fnuc) over-time (A, C), and corresponding statistics ± SEM (B, D) representing ΔΨmito of TMRM-loaded MMTV-PyMT WT and BK-KO (A, B) and MDA-MB-453 cells (C, D) under basal conditions (A, C, upper images) and upon administration of FCCP for mitochondrial depolarization (A, C lower images). (E – J) [ATP]mito dynamics ± SEM over-time of MMTV-PyMT WT and BK-KO cells (E), MDA-MB-453 cells (G) and MCF-7 cells (I) in response to extracellular glucose removal (left panels) or upon administration of Oligomycin-A (right panels). F, H and J show changes of [ATP]mito induced by glucose removal to Oligomycin-A administration ± SEM, under control conditions, or in the presence of paxilline or iberiotoxin (F, H), or upon expression of BKCaRFP or BKCa-DECRFP (J). (K – M) Basal mitochondrial H2O2 concentrations ± SEM of MMTV-PyMT WT (K, left), BK-KO (K, right), MDA-MB-453 (L) and MCF-7 cells (M), either under control conditions, in the presence of paxilline or iberiotoxin (K, L), or upon expression of BKCaRFP or BKCa-DECRFP (M). (N, P) Representative fluorescence wide-field images (left) and corresponding statistics ± SEM (right) of MMTV-PyMT WT (N, left images and red bars) and BK-KO cells (N, right images and black bars) or MDA-MB-453 cells (P) incubated with 2-NBDG, either in the absence (upper images) or presence of FCCP (lower images). (O, Q) Average ± SEM of FCCP induced change in 2-NBDG uptake of MMTV-PyMT WT (O, left) and BK-KO cells (O, right), or MDA-MB-453 cells (Q) either under control conditions, or in the presence of paxilline or iberiotoxin. Values above 1 indicate that mitochondria prevent, values below 1 that mitochondria support glucose uptake. N (independent experiments) / n (cells analyzed) = A, B: 4/75 WT ctrl, 4/90 WT + PAX, 4/86 WT + IBTX, 4/91 BK-KO ctrl, 4/89 BK-KO + PAX, 4/100 BK-KO + IBTX, C, D: 4/113 ctrl, 4/97 + PAX, 4/103 + IBTX, E, F: [-Glucose]: 8/55 WT ctrl, 6/45 WT + PAX, 7/27 WT + IBTX, 8/65 BK-KO ctrl, 6/57 BK-KO + PAX, 7/28 BK-KO + IBTX, [+ Oligomycin-A]: 11/52 WT ctrl, 7/53 WT + PAX, 7/34 WT + IBTX, 8/87 BK-KO ctrl, 6/35 BK-KO + PAX, 5/45 BK-KO + IBTX. G, H: [-Glucose]: 5/14 ctrl, 3/13 + PAX, 5/13 + IBTX, [+ Oligomycin-A]: 5/33 ctrl, 3/21 + PAX, 8/27 + IBTX, I, J: [-Glucose]: 6/48 ctrl, 5/23 + BK RFP, 5/20 + BK -DECRFP, [+ Oligomycin-A]: 5/27 ctrl, 5/23 + BK RFP, 5/37 + BK -DECRFP, K: 3/33 WT ctrl, 4/51 WT + PAX, 4/54 WT + IBTX, 4/55 BK-KO ctrl, 4/51 BK-KO + PAX, 4/54 BK-KO + IBTX, L: 4/31 ctrl, 4/39 + PAX, 4/31 + IBTX, M: 4/29 ctrl, 4/17 + BKCaRFP, 4/21 + BKCa-DECRFP, N – Q: 4 for all. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (B, D, F, H, J, K, O), Brown-Forsythe and Welch ANOVA test followed by Games-Howell’s MC test (L, M), Mann-Whitney test (N), Unpaired t-test (P) or Welch’s t-test (Q). #p≤0.05, †p≤0.01, ‡p≤0.001, to respective WT condition, Mann-Whitney test (B, F, + PAX and + IBTX in K, ctrl in O), Unpaired t-test (ctrl in K, + PAX and + IBTX in O).
BKCa activity is present in the inner mitochondrial membrane (IMM) of BCCs.
(A) Representative BKCa single-channel recordings of the IMM of mitoplasts isolated from MDA-MB-453 cells using a symmetric 150/150 mM isotonic KCl solution containing 100 µM Ca2+ at voltages ranging from -80 to +80 mV as indicated in the panel. (B) Open probability analysis of mitoBKCa at different voltages received from experiments as performed in (A). N = 8. (C) Single-channel currents of the IMM of mitoplasts isolated from MDA-MB-453 cells recorded using a voltage ramp protocol ranging from −150 to +150 mV. Above and below the ramp are enlarged excerpts of the records shown in rectangles. (D) Current-voltage (I-V) plot based on single-channel recordings of MDA-MB-453 cells as performed in a, using a symmetric 150/150 mM KCl isotonic solution containing 100 µM Ca2+. N = 11. (E, F) Representative single-channel recordings of the IMM of mitoplasts isolated from MDA-MB-453 cells (E) and corresponding open probabilities at +40 mV in a symmetric 150/150 mM KCl isotonic solution under control conditions (100 μM Ca2+), after reducing Ca2+ to 1 μM, re-addition of 100 μM Ca2+ and finally after application of 5 μM paxilline in the presence of 100 μM Ca2. Data in (F) show average ± SEM. *p≤0.05, **p≤0.01 using Friedmann test followed by Dunn’s multiple comparison test, n =7. (G) Pie chart displaying the percentage of mitoBKCa channel currents (green) possessing a conductance of ∼210 pS, versus the total number of patch-clamp experiments performed using mitoplasts isolated from MDA-MB-453 cells (upper left), MMTV-PyMT WT cells (upper right), MCF-7 cells (lower left) and MMTV-PyMT BK-KO cells (lower right). Black segments represent empty patches, bright- and dark grey fraction demonstrate percentage of channels possessing smaller conductances of ≤100 pS and ≤150 pS, respectively. All recordings were low-pass filtered at 1 kHz. “c“ and “o” indicate the closed- and open state of the channel, respectively.
BKCa-DEC expression contributes to the metabolic remodeling and growth of murine and human BCCs and is present in primary tumor samples.
(A) Schematic representation of the fate of glucose in glycolysis. The tricarboxylic acid (TCA) cycle or lactate secretion via monocarboxylate transporters (MCT) can be inhibited, either using NaN3 or BAY-8002. GLUT: Glucose transporter, HKs: Hexokinases. (B) Representative cytosolic lactate concentration ([LAC]cyto) of a MMTV-PyMT WT cell over-time in response to administration or removal of NaN3 and BAY-8002 at time points indicated. Dashed lines indicate slopes taken for assessment of the “Warburg index. (C – G) Average Warburg indices ± SEM of MMTV-PyMT WT (C, F, left), MMTV-PyMT BK-KO (C, F, right), MDA-MB-453 cells (D, G) and MCF-7 cells (E) calculated from the experiments as shown in (B), either under control conditions, in the presence of paxilline or iberiotoxin (C, D), upon expression of BKCaRFP or BKCa-DECRFP (E), or upon cell treatment with a scrambled siRNA (siScrbl), or siRNA against a common BKCa sequence targeting all known splice variants (siBK), or a siRNA specifically designed to knockdown BKCa-DEC (siDEC) (F, G). (H, I) Normalized MTT absorbance over-time of MMTV-PyMT WT (H, left) and BK-KO cells (H, right), and MDA-MB-453 cells (I), either under control conditions, or in the presence of paxilline or iberiotoxin. J, Representative images and corresponding statistics of colony formation assays using MMTV-PyMT WT or BK-KO cells in the presence or absence of O2. (K – N) mRNA expression of BKCa and BKCa-DEC as performed by Nanostring analysis of 551 BC patient samples. (K) Log2 expression counts of BKCa and BKCa-DEC. The threshold for positive expression level was set to log2 = 5.5 (dashed line). (L) Log2 expression counts of BKCa-DEC blotted over the log2 expression counts of BKCa. 10 of the 551 patient samples showed expression of BKCa-DEC above the threshold of log2 = 5.5 (dashed line), whereas 541 patient samples were BKCa-DEC negative. (M) Correlation of the log2 expression counts of BKCa-DEC positive samples with the log2 expression counts of BKCa in the primary human BC material. (N) Summarizing scheme of BKCa in cancer cell homeostasis. N (independent experiments)/ n (cells analyzed) = C: 4/26 WT ctrl, 6/28 WT + PAX, 4/39 WT + IBTX, 4/17 BK-KO ctrl, 5/18 BK-KO + PAX, 4/27 BK-KO + IBTX, D: 7/29 ctrl, 5/13 + PAX, E: 5/27 ctrl, 5/20 + BKCaRFP, 7/26 BKCa-DECRFP, F: 5/22 WT siScrbl, 5/28 WT siBK, 4/26 WT siDEC, 5/24 BK-KO siScrbl, 5/24 BK-KO siBK, 5/29 BK-KO siDEC, G: 5/21 siScrbl, 5/22 siBK, 5/19 siDEC, H – J: 4 for all. *p≤0.05, **p≤0.01, ***p≤0.001, Kruskal-Wallis test followed by Dunn’s MC test (C, E, F, G, I), One-Way ANOVA test followed by Tukey’s MC test (H) or Mann-Whitney test (D). †p≤0.01, ‡p≤0.001 compared to respective WT condition, Unpaired t-test (ctrl, C, J) or Mann-Whitney test (+ IBTX, C, F).
