Ani9, A Novel Potent Small-Molecule ANO1 Inhibitor with Negligible Effect on ANO2
Fig 4
A) Intracellular calcium concentration was measured using Fluo-4 in FRT cells. The cells were pretreated with the indicated concentrations of Ani9 for 20 min and then 100 μM ATP, an agonist of P2Y receptor, was applied. B) Apical membrane current was measured in FRT cells expressing CFTR. CFTR was activated by 20 μM forskolin and inhibited by 10 μM CFTRinh-172. C) ENaC activity was measured in T84 cells. ENaC was fully blocked by 100 μM amiloride. D) VRAC activity was measured in LN215 Cells expressing endogenous VRAC and a halide sensor YFP. VRAC activity was inhibited by the indicated concentrations of ANO1 inhibitors and 10 μM DCPIB, a potent VRAC inhibitor. E, F) Whole-cell VRAC current was recorded at a holding potential at 0 mV and was pulsed to voltages between ± 100 mV (in steps of 20 mV) in the absence and presence of 10 μM DCPIB (E) or 1 μM Ani9 (F) in LN215 cells. G) Effect of Ani9 on ANO1 activation by Eact in FRT-ANO1 cells. 10 μM Ani9 was pretreated for 20 min and ANO1 was activated by 10 μM Eact. The remaining ANO1 current was inhibited by 10 μM T16Ainh-A01. (right) Summary of peak current (mean ± S.E., n = 3–4). H) Ani9 reversibility. After vanishment of 100 μM ATP-induced ANO1 current, the cells were washed six times for 5 min each and then ANO1 was activated by 10 μM Eact. (right) Summary of peak current (mean ± S.E., n = 3–4). **P < 0.01, ***P < 0.001.