Fig. 1.

Identification of Ca²⁺ signaling cascade potentiators by high-throughput screening. (A) Scheme of the screening assay. FRT cells with co-expression of the TMEM16A Cl⁻ channel and HS-YFP were preincubated for 20 min with compounds in 96-well microplates. For the assay, the microplate reader continuously recorded cell fluorescence before and after addition of a saline solution containing I⁻ instead of Cl⁻ plus a submaximal UTP concentration (0.25 µM). TMEM16A channel activation by UTP resulted in I⁻ influx and HS-YFP quenching. Presence of an active compound in the well was detected by faster and larger quenching. (B) Detection of active compounds by HS-YFP assay. Representative traces (left) and summary of data (right) obtained for indicated compounds. The scatter dot plot reports activity as cumulative fluorescence quenching (CFQ). *P <0.05; **P <0.01; ***P <0.001 vs. vehicle (ANOVA with Dunnett’s post-hoc test). (C) Representative traces (left) and summary of data (right) from short-circuit current (Isc) recordings on FRT cells with stable expression of TMEM16A. Cells were briefly pre-incubated with indicated compounds (10 µM) or vehicle and then stimulated with 0.25 µM UTP (on the apical side) to induce TMEM16A-dependent Cl⁻ transport. The scatter dot plot reports the value of maximal UTP effect. The current activated by UTP is significantly enhanced by ARN7149, ARN11391, and ARN4550 compared to vehicle. **P <0.01; ***P <0.001 (ANOVA with Dunnett’s post-hoc test). (D) Dose-response of ARN7149, ARN11391 and ARN4550 by HS-YFP assay in FRT cells expressing TMEM16A.