Fluorescent membrane potential assay for drug screening on Kv10.1 channel: identification of BL-1249 as a channel activator

Figures

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  FIGURE 1. FLIPR® membrane potential assay for Kv10.1 channel activity. (A) Membrane potential recordings in HEK-WT (cyan) and HEK-Kv10.1 (blue) cells using the FLIPR®-blue formulation. Arrow indicates when the high potassium (60 mM) stimulus was added to cells. HEK-Kv10.1 cells without any dye (FMP(-); black line) showed no fluorescent signal. The solid traces and faded colors show the mean ± SEM, respectively. (B) First derivative of the fluorescents signal showed that high potassium solutions induce faster signals in HEK-Kv10.1 than HEK-WT cells. (C) Comparison of the maximum fluorescence change in the different cells evaluated.

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  FIGURE 2. FLIPR® membrane potential for HEK-Kv10.1 cells. (A) Summary bar graph of the fluorescence responses in HEK-Kv10.1 cell in the presence of different small molecules. Data were normalized to the control wells. Each bar represents the mean ± SEM of at least three independent experiments. (B–D) Representative recordings of the effect of fluoxetine, miconazole, and BL-1249 on the Kv10.1-FMP responses, respectively. In D, the insert represents the changes in the basal fluorescence in control (black) and the presence of BL-1249.

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  FIGURE 3. Inhibitory effect of fluoxetine on Kv10.1 channels. (A,B) Representative traces of a family of Kv10.1 currents in control (black) and in the presence of fluoxetine (100 μM; orange), respectively. (C) I-V relationship of Kv10.1 currents in control (black circles) and the presence of fluoxetine (100 μM; orange circles) (n = 5). (D) Dose-inhibition curves of fluoxetine on Kv10.1 currents (n ≥ 3 for each point). The solid line represents the fit to the Hill equation.

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  FIGURE 4. Effect of miconazole nitrate (MN) on Kv10.1 currents. (A) The temporal course of the inhibitory effect of MN (1, 10, and 100 µM) on Kv10.1 currents. The current traces for the time points marked with filled dots are presented in B. (B) Kv10.1 recordings in control (black) and in the presence of 1 (teal), 10 (purple), and 100 (pink) µM of MN (C) I-V relationship of Kv10.1 currents in control (black circles), the presence of MN (100 μM; teal circles), and during the washout (empty circles) (n = 5). (D) Dose-inhibition curves of MN on Kv10.1 currents (n ≥ 3 for each point). The solid line represents the fit to the Hill equation.

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  FIGURE 5. BL-1249 (BL) exhibits opener activity on Kv10.1 currents. (A) The temporal course of the opener effect of BL (100 µM) on Kv10.1 currents. The current traces for the time points marked with filled dots are presented in B. (B) Kv10.1 recordings in control (black), in the presence of BL (100 µM), and during the washout (gray). (C) Summary of the dose-dependent effect of BL on the Kv10.1 current. (D) Conductance-voltage relationship of the Kv10.1 channels in control and the presence of BL. The solid line represents the fit to the Boltzmann equation.

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  FIGURE 6. BL-1249 hyperpolarizes the resting membrane potential of HEK-Kv10.1 cells. (A,B) Current-clamp recording of RMP of HEK-Kv10.1 cells in the presence of DMSO and BL (100 µM), respectively. (C,D) Effects on the RMP of HEK-Kv10.1 cells recorded in the presence of DMSO (n = 4) and BL (n = 7), respectively. (E,F) Average effect of DMSO and BL on the RMP of HEK-Kv10.1 cells. Above, data represent the shift of RMP in each cell concerning the control condition (dotted line) obtained before applying every compound. Below is a summary histogram of the average change of the RMP.

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  FIGURE 7. BL-1249 does not affect the endogenous potassium currents or RMP of HEK-WT cells. (A, B) representative family of endogenous K+ currents in HEK-WT cells in control and the presence of 100 μM BL-1249, respectively. (C) I-V relationship of HEK-WT currents in control (black circles) and the presence of BL-1249 (100 μM; red circles) (n = 10). (D) Representative current-clamp recording of RMP in HEK-WT cells in the presence of BL (100 μM). (E) The average effect of BL on the RMP of HEK-WT cells in control, in the presence of BL, and during the washout (n = 6).

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  FIGURE 8. The opener activity of BL-1249 on Kv10.1 currents is obliterated in the presence of fluoxetine. (A) The time course of the activity of fluoxetine (30 μM; Fluox), fluoxetine plus BL-1249 (100 μM; BL), and BL on Kv10.1 currents. The current traces for the time points marked with filled dots are presented in B. (B) Kv10.1 recordings in control (black), in the presence of fluoxetine (red), fluoxetine plus BL (blue), BL (green), and during the washout (purple). (C) Summary of each treatment on Kv10.1 currents. Data are normalized to the control condition.

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  FIGURE 9. Alignment of the S6 segment of the human Kv10.1 and Kv11.1 channels. Conserved aromatic amino acids are shaded in gray. Sequence numbering is shown on the right. Arrow indicates the position of residue F656 in Kv11.1.