Figure 2.

Measurements of ANO6 Ca²⁺-activated ion channel and phospholipid scramblase activities

(A–C) The inhibitory effect of A6-001 on the ANO6 ion channel activity was analyzed using a whole-cell patch clamp in HEK 293T cells expressing ANO6. The pipette solution contained 10 μM free Ca²⁺. Voltage ramps spanning a range of −100 to +100 mV were delivered from a holding potential of −60 mV every 20 s (A). The current-voltage (I-V) relationships at indicated times (1–5) with increasing concentrations of A6-001 are shown in (B). The numbers in parentheses represent the time points to measure I-V relationships. The inward currents in (A) represent ANO6 tail currents. The half-maximal inhibitory concentration (IC₅₀) of A6-001 on the ANO6 ion channel activity is depicted in (C) (n = 4 at each concentration).

(D–F) The effects of ANO6 inhibitors on the surface exposure of phosphatidylserine (PS) were assayed in FRT-ANO6 cells. Cells were pretreated with compounds for 10 min, before ANO6 was activated with 10 μM ionomycin. Exposed PS was stained with Lact-C2-GFP. Representative fluorescence images of A6-001 (10 μM), A6-004 (10 μM), abamectin (10 μM), and ivermectin (10 μM) are shown in (D) (scale bars: 10 μm), and fluorescence intensity values calculated from Lact-C2-GFP images are summarized in (E) (mean ± SEM; n = 6). The dose responses of A6-001 on ANO6 PS scramblase inhibition are analyzed in (F) (mean ± SEM; n = 6). Representative fluorescence images of A6-001 dose response are shown in Figure S2H.

(G and H) The inhibitory effect of A6-001 on PS scrambling was assayed using the four-quadrant dot blot FACS analysis in Jurkat cells, some of which were transfected with hANO6-expressing plasmids using a NEPA21 Super Electroporator (Jurkat-ANO6). Ionomycin evoked an increase in the Lact-C2-positive and propidium iodide (PI)-negative cell population (Q3), which represented the PS-exposed and non-apoptotic cells, respectively. The ionomycin-induced Q3 increase was augmented considerably in cells expressing exogenous ANO6. A6-001 (10 μM) reversed the Q3 increases in Jurkat and Jurkat-ANO6 cells. Representative FACS analyses are shown in (G), and a summary of the multiple experiments is presented in (H) (mean ± SEM; n = 5). ^∗p < 0.05 and ^∗∗p < 0.01: differences from lane 2.

Data were analyzed using one-way analysis of variance, followed by Tukey’s multiple comparison test. See also Figure S2.