Fig 1.
Identification of novel inhibitors of ANO1.
A) Chemical structures of ANO1 inhibitors. B) Apical membrane current measured in ANO1 expressing FRT cells in the presence of a transepithelial chloride gradient. Representative current traces showing Ani1, Ani7 and Ani9-induced ANO1 inhibition at the indicated concentrations. ANO1 was activated by 10 μM Eact, a specific activator of ANO1. The dashed gray line shows a control trace.
Fig 2.
ANO1 inhibition by analogs of Ani9.
Structure-activity analysis of Ani9 analogs. IC50 values were determined from fluorescence plate reader assay.
Fig 3.
Ani9, a potent inhibitor of ANO1.
A-C) Apical membrane current was measured in ANO1-expressed FRT cells. The indicated concentrations of ANO1 inhibitors, T16Ainh-A01, MONNA and Ani9 were applied 20 min prior to ANO1 activation by 100 μM ATP. D) Summary of dose responses (mean ± S.E., n = 4–6).
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.
Fig 5.
Patch-clamp analysis of ANO1 inhibition by Ani9 in FRT-ANO1 cells.
A) Whole-cell ANO1 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 50 nM, 100 nM and 1 μM Ani9. ANO1 was activated by 100 μM ATP. B) Current/voltage (I/V) plot of mean currents at the middle of each voltage pulse. C) The bar graphs summarize the current density data measured at + 80 mV (mean ± S.E., n = 4–6). ***P < 0.001.
Fig 6.
Effect of Ani9 on ANO2 activity.
A-C) YFP Fluorescence measurement showing inhibition of mouse ANO2 activity by Ani9, T16Ainh-A01 and MONNA in FRT cells expressing ANO2 and a halide sensor YFP. The indicated concentrations of the inhibitors were applied 20 min prior to ANO2 activation by 100 μM ATP. D) Apical membrane current was measured in FRT-ANO1cells. 1 μM Ani9, 10 μM T16Ainh-A01 and 10 μM MONNA were applied 20 min prior to ANO1 activation. E) Apical membrane current was measured in FRT-ANO2 cells. 1 μM Ani9, 10 μM T16Ainh-A01 and 10 μM MONNA were applied 20 min prior to ANO2 activation. F) Summary of peak currents of ANO1 and ANO2 (mean ± S.E., n = 4). G) Representative current traces showing Ani9-induced ANO2 inhibition at the indicated concentration. H) Summary of dose responses of ANO1 and ANO2 (mean ± S.E., n = 4–6).
Fig 7.
Effect of Ani9 on endogenous CaCCs in PC3, Capan-1 and NHNE cells.
A) Immunoblot of ANO1 protein in FRT, FRT-ANO1, PC3, Capan-1 and NHNE cells. Blots shown are representative of experiments performed three times. B-C) Effect of Ani9 on CaCCs activity was measured in PC3 and Capan-1 cells expressing a halide sensor YFP. The indicated concentrations of Ani9 were applied 20 min prior to CaCCs activation by 100 μM ATP. D) Summary of dose response (mean ± S.E., n = 6). E) Effect of Ani9 on CaCCs activity in IL-4 treated (10 ng/mL; 48h) NHNE cells. CaCCs currents were induced by 100 μM ATP and then the indicated concentrations of Ani9 were applied. The remaining CaCCs currents were abolished by 100 μM tannic acid (TA). (right) Summary of dose response (mean ± S.E., n = 4).