Characterization of idebenone, a TMEM16A inhibitor.
(A) Apical membrane currents were measured in FRT-ANO1 cells. Idebenone was added 10 min prior to ANO1 activation by 100 μM ATP. (B) Summary of dose-response (mean ± S.E., n = 3–4). (C) Intracellular calcium concentration was measured using Fluo-4 in HT-29 and FRT cells. 30 μM idebenone (IDE), miconazole (MCZ) and plumbagin (PLB) were pretreated for 20min and then 100 μM ATP was applied. (D) Effect of idebenone on CFTR chloride channel activity was measured in FRT cells expressing human wild-type CFTR. CFTR was activated by 20 μM forskolin and inhibited by 10 μM CFTRinh-172. (E) Effect of idebenone on mouse ANO2 (mANO2) was measured in FRT-mANO2 cells. (F) Effect of Coenzyme Q10 (CoQ10) on ANO1 channel activity was observed in FRT-ANO1 cells. 100 μM CoQ10 was pretreated for 20min and then 100 μM ATP was applied. (right) Summary of peak current (mean ± S.E., n = 3–4). (G) Effect of idebenone on ANO1 activation by Eact in ANO1-expressing FRT cells. 100 μM idebenone (gray line) was pretreated for 20min and ANO1 was activated by 10 μM Eact. The remaining ANO1 currents were inhibited by T16Ainh-A01. (right) Summary of peak current (mean ± S.E., n = 3). (H) Idebenone reversibility. After vanishment of 100 μM ATP-induced ANO1 current, the cells were washed three times for 5 min each and then ANO1 was activated by 10 μM Eact. (right) Summary of peak current (mean ± S.E., n = 3). **P < 0.01, ***P < 0.001, Students’ unpaired t-test.
