Tamoxifen metabolites acting via BKCa orchestrate the dynamics of K+ and Ca2+ in breast cancer cells

Figures

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  Figure 1. BKCa expression in human BC biopsies and electrophysio-/pharmacological properties of human BC cell BKCa channels. mRNA expression levels of (A) KCNMA1 and selected cancer-associated K+ channels from human BC biopsies (partly adapted from (7)) and (B) KCNMA1 in human BC cell lines hBKCalow, hBKCamedium and hBKCahigh. C, current-density-voltage relationships of hBKCalow, hBKCamedium and hBKCahigh compared at +120 mV (data of selected voltage was adapted from the recordings in D–F). D–I, representative whole-cell current recordings (left) and current density-voltage relationships (right) of hBKCalow, hBKCamedium and hBKCahigh BC cells, (D, E, F) before and during paxilline (PAX), (G) NS11021, (H) 4-OH-TAM, and (I) endoxifen treatment. Data represents average ± SD. n(biopsies) = (A) 551; n(independent experiments) = (B) 6; n(cells) = (C) 7-9, (D) 7, (E) 7, (F) 9, (G) 11, (H) 8, (I) 8, with ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. A and C, Kruskal-Wallis test followed by Dunn’s multiple comparisons test. B, one-way ANOVA followed by Tukey’s multiple comparisons test. D–I two-way ANOVA followed by Šídák‘s multiple comparisons test. Statistical values, probe and primer sequences are…

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  Figure 2. TAM+M trigger cytosolic K+ dynamics in BC cells independently of ER. (A, B, C, F, and G), K+ FRET-ratio signals obtained by live-cell imaging of cytosolic K+ concentrations ([K+]i) over time of hBKCalow (ER+ or ER-), hBKCamedium (ER+) and hBKCahigh (ER-) BC cells and (A–G) corresponding bar charts with individual data points for statistical analysis, which is based on the difference of the basal (R0) to the minimal (R) K+ FRET/CFP ratio signals and expressed as ΔMin(R/R0). Alterations of [K+]i were induced by bath-administration of (A, B) BKCa activator NS11021 (hBKCalow from A adapted in B), active TAM metabolites (C) 4-OH-TAM and (D, E, G) endoxifen or (F) E2 following 10 min of FRET recording at basal conditions. ER+ hBKCalow cells expressed in addition either (A, G) RFP (+RFP), RFP-tagged BKCa (+BKCa-RFPrescue) or RFP-tagged “pore-death” BKCaG354S (+BKCaG354S) as indicated. FRET-ratio signal responses are displayed for (C, E, G) 1 μM 4-OH-TAM or endoxifen and (F) 1 μM E2. Data represents average ± SD. n(independent experiments) = (A) 4-5, (B) 5-7, (C) 3-6, (D) 4-6, (E) 4-6, (F) 3-4, (G) 5 with ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (A, B, E, G) One-way ANOVA followed by Tukey‘s…

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  Figure 3. TAM+M-mediated effects on murine and human BC cell K+ homeostasis require functional BKCa channels. A–D, FRET-ratio signals and corresponding statistics of cytosolic K+ ([K+]i) dynamics over time in murine MMTV-PyMT BKCa WT (mBKCaWT) and KO (mBKCaKO) and human hBKCalow and hBKCahigh BC cells. Live-cell K+ imaging was performed prior and after bath-application of (A) endoxifen, (B) 4-OH-TAM, (A and C) PAX+endoxifen and (D) PAX+4-OH-TAM. C, PAX+endoxifen and (D) PAX+4-OH-TAM treated hBKCalow and hBKCahigh BC cells were compared with 1 μM 4-OH-TAM and 1 μM endoxifen (Fig. 2, C and D) respectively. Data represents average ± SD. n(isolated tumors) = (A and B) 4-6 and n(independent experiments) = (C) 3-5, (D) 4-6 with ∗p ≤ 0.05 and ∗∗∗p ≤ 0.001. A, one-way ANOVA followed by Tukey‘s multiple comparisons test. (B) Unpaired t test. C and D two-way ANOVA followed by Šídák‘s multiple comparisons test. Statistics are described in detail in Table S3.

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  Figure 4. TAM+M-induced BKCa activation elevates [Ca2+]i. Fluorescence emission ratio signals over time and area under the curve (AUC) statistics of hBKCalow, hBKCamedium, and hBKCahigh BC cells loaded with the ratiometric Ca2+ dye Fura-2 in presence of (A) 4-OH-TAM or (B) endoxifen. Fluorescence emission ratio traces (F340/F380) of hBKCalow (Fig. S3A) were compared with hBKCahigh (Fig. 4C). F340/F380 nm emission ratio for Fura-2 loaded (C) hBKCahigh BC cells in response to endoxifen in addition with further treatment by (from left to right) BKCa inhibitor PAX, BAPTA AM (BAPTA) for intracellular Ca2+ depletion, EGTA-buffered extracellular Ca2+-free solution, and TRP/C, STIM/ORAI1 channel blocker SKF-96365 (SKF). D, analysis of the area under the curve (AUC) of Fura-2 loaded hBKCahigh BC cells under recording conditions specified in (C). E, fura-2 fluorescence emission ratio signals (F340/F380 nm) in response to Ca2+ changes over time and statistics obtained from monitoring hBKCahigh BC cells after pre-treatment with endoxifen, PAX+endoxifen, EGTA+endoxifen, SKF+endoxifen (from left to right) followed by extracellular ATP application to provoke emptying of intracellular Ca2+ stores. F,…

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  Figure 5. The TAM+M-BKCa channel axis, by modulating [K+]i, initiates intracellular Ca2+ signals and a depolarized ΨPM in BC cells. A, FRET ratio signals of cytosolic K+ changes over time and (B) statistical presentation of minimal (R) to basal (R0) ratio (ΔMin) monitored in hBKCahigh BC cells in presence of endoxifen (Fig. 2D, 1 μM endoxifen), depleted intracellular Ca2+ by BAPTA AM (BAPTA), Ca2+-free, EGTA-buffered extracellular conditions, and TRP/C, STIM/ORAI1 channel block by SKF-96365 (SKF) (from left to right). C, representative whole-cell current tracings (left) and current density-voltage curves (right) of hBKCahigh BC cells in presence of EGTA before and after endoxifen administration. D, representative fluorescence images (basal (left), endoxifen (middle)) and fluorescence intensity signals recorded over time (right) from hBKCalow and hBKCahigh BC cells loaded with the membrane potential (ΨPM)-sensitive dye DiBAC4(3) and administration of endoxifen. Scale bar = 10 μm. E fluorescence intensity signals of hBKCahigh BC cells loaded with ΨPM-sensitive dye DiBAC4(3) in the presence of PAX+endoxifen, BAPTA AM+endoxifen and EGTA+endoxifen. F, quantification of endoxifen-induced maximal…

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  Figure 6. TAM+M enhances single channel open probabilities independent of Ca2+ levels. A and B, representative single channel recordings (left) and corresponding bar charts (right) of hBKCahigh BC cells at negative and positive voltages (-50 and +50 mV) using a symmetric isotonic KCl solution containing either (A) Ca2+-free or (B) physiological Ca2+ levels. 1 μM endoxifen significantly increased BKCa open probability (NPO) under all at investigated conditions. Data represents average ± SD. n (cells) = (A) 8 and (B) 10 with ∗p ≤ 0.05 and ∗∗p ≤ 0.01. (A) Wilcoxon test. (B) Paired t test. Statistical values are described in detail in Table S6.

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  Figure 7. Molecular modelling of TAM+M interaction with hBKCa. Results of docking simulation of TAM+M into (A, B) extracellular binding pocket “WDF” and (C, D) pore-interacting binding pocket “TSYF” in (A, C) tetrameric arrangement of α-subunits and (B, D) magnified regions of putative binding modes in cryo-EM structures of Ca2+-bound BKCa (PDB 6V38) after induced fit docking. The high docking scores indicate strong hydrophobic, polar and π-π interactions at (A, B) segments S0-S5 with the amino acids W22, D261 and F252 and (C, D) S1, S5-S6 with T298, S134, Y274, F131. Color code: Segments, light blue; K+, red sphere; Endoxifen, yellow; 4-OH-TAM, green. E, summarizing scheme of BKCa-mediated alterations in K+ and Ca2+ homeostasis due to TAM+M stimulating BKCa channel activation. (1) TAM+M provoke BKCa channel activity, (2) BKCa-dependent K+ dynamics lead to an increased extracellular K+ ([K+]ex) and a lower [K+]i; Ca2+ entry through (3a) TRP/C-like Ca2+ channels in the PM and (3b) by its release from intracellular stores such as the endoplasmic reticulum results in a cytosolic Ca2+ accumulation ([Ca2+]i) in BC cells. This increase in [Ca2+]i in turn causes (4a) voltage- and (4b)…