The Natural Flavone Acacetin Blocks Small Conductance Ca2+-Activated K+ Channels Stably Expressed in HEK 293 Cells

Figures

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  FIGURE 1. Effect of acacetin on SKCa1 channel stably expressed in HEK 293 cells. (A) SKCa1 current was activated in a representative cell expressing human KCNN1 by 200-ms step voltages between –70 and +80 mV from a holding potential of –80 mV in the absence and presence of 10 μM acacetin. (B) Current–voltage (I–V) relationships of SKCa1 current were recorded in a typical experiment with a 3-s voltage ramp from –100 to +80 mV in the absence and presence of 10 μM acacetin. (C) Concentration–response relationship of acacetin for inhibiting SKCa1 current (+80 mV) was fitted to a Hill equation to obtain IC50 value of acacetin.

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  FIGURE 2. Effect of acacetin on SKCa2 channel stably expressed in HEK 293 cells. (A) SKCa2 current was activated in a representative cell expressing rat KCNN2 by 200-ms step voltages between –70 and +80 mV from a holding potential of –80 mV in the absence and presence of 10 μM acacetin. (B) Current–voltage (I–V) relationships of SKCa2 current were recorded in a typical experiment with a 3-s voltage ramp from –100 to +80 mV in the absence and presence of 10 μM acacetin. (C) Concentration–response relationship of acacetin for inhibiting SKCa2 current (+80 mV) was fitted to a Hill equation to obtain IC50 value of acacetin.

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  FIGURE 3. Effect of acacetin on SKCa3 channel stably expressed in HEK 293 cells. (A) SKCa3 current was activated in a representative cell expressing human KCNN3 by 200-ms step voltages between –70 and +80 mV from a holding potential of –80 mV in the absence and presence of 10 μM acacetin. (B) Current–voltage (I–V) relationships of SKCa3 current were recorded in a typical experiment with a 3-s voltage ramp from –100 to +80 mV in the absence and presence of 10 μM acacetin. (C) Concentration–response relationship of acacetin for inhibiting SKCa3 current (+80 mV) was fitted to a Hill equation to obtain IC50 value of acacetin.

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  FIGURE 4. Effect of acacetin on WT and mutant SKCa3 channel currents expressed in HEK 293 cells. (A) Current–voltage (I–V) relationships of SKCa3 WT current, H490N, S512T, H521N were recorded in typical experiments with a 3-s voltage ramp from –100 to +80 mV in the absence and presence of 10 μM acacetin. (B) Percent values of 10 μM acacetin for inhibiting SKCa3 WT current (n = 11, H490N current (n = 7, ∗∗P < 0.01 vs. WT), S512T current (n = 7, ∗∗P < 0.01 vs. WT), H521N current (n = 7, P > 0.05 vs. WT), or A537V current (n = 6) at +80 mV.

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  FIGURE 5. IC50 values of acacetin for inhibiting SKCa3 WT, H490N, S512T, H521N, or 537V current in HEK 293 cells. (A) Concentration–response relationship curves of acacetin for inhibiting SKCa3 WT current (+80 mV), H490N, S512T, H521N, or 537V (n = 6–9 for each concentration) was fitted to a Hill equation to obtain IC50 values of acacetin. (B) Schematic graph showing the putative binding sites of acacetin at H490, S512, and also H521 in the P-loop helix of human SKCa3 channels.

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  FIGURE 6. Effect of acacetin on IKCa channel stably expressed in HEK 293 cells. (A) IKCa current was activated in a representative cell expressing human KCNN4 by 200-ms step voltages between –70 and +70 mV from a holding potential of –80 mV in the absence and presence of 10 or 30 μM acacetin. (B) Current–voltage (I–V) relationships of IKCa current were recorded in a typical experiment with a 3-s voltage ramp from –90 to +70 mV in the absence and presence of 10 and 30 μM acacetin. (C) Percent values of acacetin (10 or 30 μM) for inhibiting IKCa current (+70 mV, n = 7, ∗P < 0.05 vs. 0 μM acacetin).

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  FIGURE 7. Effect of acacetin on BKCa channel stably expressed in HEK 293 cells. (A) BKCa current was activated in a representative cell expressing human KCNMA1 by 200-ms step voltages between –70 and +70 mV from a holding potential of –80 mV in the absence and presence of 3, 10, or 30 μM acacetin. (B) Current–voltage (I–V) relationships of SKCa3 current were recorded in a typical experiment with a 3-s voltage ramp from –90 to +70 mV in the absence and presence of 10 and 30 μM acacetin. (C) Percent values of acacetin (3, 10, or 30 μM) for increasing BKCa current (+70 mV, n = 7, ∗P < 0.05 vs. 0 μM acacetin).