Figure 5. BK_Ca activity is present in the inner mitochondrial membrane (IMM) of BCCs.

(A) Representative BK_Ca 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 mitoBK_Ca 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 mitoBK_Ca 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.

Figure 5—figure supplement 1. BK_Ca is present in the inner mitochondrial membrane of MMTV-PyMT WT and MDA-MB-453 cells.

(A – C) Graphs show representative BK_Ca single-channel recordings of the inner mitochondrial membrane of mitoplasts isolated from MMTV-PyMT WT cells using a symmetric 150/150 mM isotonic KCl solution, either containing 100 µM Ca²⁺ (A), 1 µM Ca²⁺ (B), or 5 µM Paxilline in the presence of 100 µM Ca²⁺ (C), at voltages ranging from –80 to +80 mV as indicated in the panels. The patch in (A) contained two BK_Ca channels. ‘c’ indicates the closed-, ‘o’ the open state of the channel. (D) Representative western blot of BK_Ca, Na⁺/K⁺ ATPase as a plasma membrane marker, cytochrome c oxidase subunit IV (COXIV) as a mitochondrial marker and thioredoxin-related transmembrane protein 1 (TMX1), a protein enriched in the ER membrane. Western blot was performed using whole-cell lysates (L), the homogenate (H), crude isolated mitochondria (C) and mitochondria after percoll gradient purification (P) of MDA-MB-453 cells. N = 3.