Figures and data
Mangostin potently activates BKα and BKα/β1 channels compared to other potassium channel representatives.
(A) Current fold change ± SEM of currents of representatives from different potassium channel families upon application of 10 µM α-Mangostin. K2P and KCa channel currents were recorded with ramp protocols from -100 mV to +50 mV and analyzed at +40 mV; Kv1.1 and Kv1.3 channel currents were evoked using a rectangle pulse to +40 mV, and hERG currents were recorded with a rectangle pulse to +60 mV followed by hyperpolarization to -120 mV, which was analyzed. Kir channels were recorded using ramp protocols from -150 mV to +50 mV, and currents were analyzed at -140mV. All measurements were made in transiently transfected HEK293 cells in physiological potassium gradients with 100 nM intracellular free Ca2+ with a holding potential of -80 mV. TWIK-1m and TWIK-2m denote channels where the retrieval motif was removed to improve membrane expression, and intracellular K+ was exchanged for Rb+ to enhance currents. (B) Representative current traces of channels that were inhibited more than 60 % by 10 µM α-Mangostin. (C) Representative current traces of BKα and BKα/β1 channels activated by 10 µM α-Mangostin (α-M). (D) Current fold change ± SEM after application of α-Mangostin, γ-Mangostin, and a dietary supplement to TREK-1, BKα and BKα/β1 channels. Currents were recorded and analyzed as in (A). (E) Representative time course of the dose-dependent activation of BKα channels by increasing concentrations of α-Mangostin (left) and the resulting dose-response-relationships for BKα and BKα/β1 channels (right). Currents were recorded and analyzed as in (A); the grey data point at 12.5 µM was not included in the Hill fit. Data and statistics see Tables S1 and S2.
Effects of α-Mangostin on BKα and BKα/β1 channel gating.
(A) Representative current traces for BKα and BKα/β1 channels before and after activation by 10 µM α-Mangostin. Cells were measured in symmetrical 140 mM bi-ionic conditions with 140 mM Cs+ as the intracellular ion and 100 nM free Ca 2+. Currents were elicited by a family of rectangle pulses from -100 mV to up to +300 mV in 20 mV increments from a holding potential of -80 mV, followed by repolarization to -50 mV to elicit inward tail currents. (B) GV-relationships for BKα and BKα/β1 channels in the basal state and activated by 10 µM α-Mangostin, derived from tail current analysis of recordings as in (A). The grey arrow illustrates the left-shift of voltage activation caused by α-Mangostin and the inset shows the slope ± SEM of the Boltzmann fits. (C) Activation and deactivation kinetics of BKα and BKα/β1 channels in the basal state and after activation by 10 µM α-Mangostin. The τ ± SEM of activation/deactivation was determined from exponential fits to the current traces at +100 mV, as shown by the green and purple colored lines in panel (A). (D) GV-relationships of BKα channels in different free Ca 2+ concentrations before and after activation by 10 µM α-Mangostin, derived from tail current analysis as above. Pulse voltages were -100 mV to +300 mV/ repolarization to -50 mV from a holding potential of -80 mV for 100 nM free Ca 2+, -120 mV to +200 mV/ repolarization to -50 mV from a holding potential of -80 mV for 1 µM free Ca 2+, and -160 mV to +100 mV/ repolarization to -80 mV from a holding potential of -120 mV for 10 µM free Ca 2+, in symmetrical 140 mM bi-ionic conditions with 140 mM Cs+ as the intracellular ion. The V½ values ± SEM before and after α-Mangostin application and the resulting shifts (Δ V½ ± SEM) are shown in the bar graphs. Data and statistics see Tables S3 – S5.
Activation mechanism of α-Mangostin.
(A) Exemplary current traces of a single BKα channel in the basal state and after activation by 10 µM α-Mangostin (1 min recordings; inset shows 1 s; O and C denote open and closed levels). Single-channel currents were recorded at +40 mV in inside-out patches from transiently transfected HEK293 cells in symmetrical potassium gradients with 100 mM free Cai2+ (n=4-7). (B) All-points histograms with Gaussian fits for the basal and α-Mangostin activated state, and bar graphs of the derived mean ± SEM open probabilities (Po) and amplitudes. The inset magnifies the open peak in the basal state. (C) Closed and open dwell time histograms with fits for channels in the basal and in the α-Mangostin activated state derived after event detection in single-channel measurements as shown in (A). (D) Normalized mean ± SEM currents of BKα channels before and after application of 3 µM or 10 µM α-Mangostin in the closed state. Channels were held closed at -80 mV and only very shortly pulsed to +60 mV after 5 min incubation to assess the current size/activation state of the first and the following pulses. Measurements were done in transiently transfected HEK293 cells in whole-cell mode in physiological potassium gradients with 100 nM Cai2+ as shown by the representative current traces to the right and steady-state currents were analyzed (grey arrow). Data and statistics see Table S6.
Investigation of the binding site of α-Mangostin in BKα channels.
(A) Competition experiment showing a reduction of the block caused by THexA in the presence of α-Mangostin. Left, representative current traces for different THexA concentrations in the presence of 10 µM α-Mangostin; middle, competition experiment analysis showing the relative current of BKα channels in different THexA concentrations in the absence and in the presence of 10 µM α-Mangostin or 100 µM BC5, which does not bind in the pore; and right, estimated IC50 values of THexA alone and in the presence of α-Mangostin or BC5. Whole-cell currents were recorded from transiently transfected HEK293 cells with a ramp protocol (-100 mV to +50 mV) in a physiological potassium gradient with 100 nM free Cai2+ and data are shown as mean ± SEM at +40 mV. (B) Molecular docking of α-Mangostin to the human BK channel structure (PDB ID 6v3g). The full-length structure (green) was reduced to the inner pore region (pink) for the docking, and the zoom-in shows the best pose for α-Mangostin with interacting residues in stick representation; green residues mark hits from the following functional assay. Protein chain B was removed for clarity. (C) Voltage of half-maximal activation (V½) before and after activation by 10 µM α-Mangostin in different pH and the resulting shifts in V½ (Δ V½). The pH was changed intra- and extracellularly. (D) Voltage of half-maximal activation (V½) before and after activation by 10 µM α-Mangostin, and the resulting shifts in V½ (Δ V½). (E) GV-relationships for the six BKα mutants in the S6 segment. (F) Voltage of half-maximal activation (V½) before and after activation by 1 µM GoSlo-SR-5-6, the resulting shifts in V½ (Δ V½), and the GV-relationships for the wildtype and two BKα mutants. GV-relationships were measured as in Fig. 2, and all data represent mean ± SEM. Data and statistics see Tables S9 – S12.
α-Mangostin activation of BK channels in physiological settings.
(A) Representative whole-cell current traces of BKα/β1 channels alone and BKα/β1 coexpressed with Cav1.2 channels before and after application of 10 µM α-Mangostin. Currents were measured in Cai2+-free conditions in a physiological potassium gradient with a family of voltage steps from -50 mV to +50 mV in 10 mV increments. The inset shows voltage activation with a family protocol up to +200 mV to show the presence of BKα/β1 channels. (B) Currents of BKα/β1 channels and BKα/β1 – Cav complexes before and after application of 10 µM α-Mangostin plotted against voltage. The last panel shows the α-Mangostin-activated currents for the range -50 to 10 mV obtained by subtracting the current before α-Mangostin application from the current after application for each potential (mean ± SEM, n=8-11 for each condition). (C) Representative contraction force recordings of aortic preparations from mice. 10 µM α-Mangostin were either applied directly to aortic preparations precontracted with 100 nM Noradrenaline (NA; top), or the precontracted preparations were incubated with 100 nM Iberiotoxin (IbTx) before α-Mangostin application and the contraction force was analyzed 10 min after α-Mangostin addition (dotted lines in recordings). The bar graph shows the normalized contraction force of preparations as mean ± SEM together with the median (orange). Data and statistics see Table S13.
