Screening Compounds for Ca2+ Channel Modulation

The effect of compounds (Ar1-Ar24) on human Ca²⁺ channels was evaluated using Fluo-4 Direct calcium channel assay kit (Life Technologies, Carlsbad, CA), according to the manufacturer’s instructions. As described earlier [31 (link),39 (link)], the 2X Fluo-4 Direct calcium reagent loading solution containing probenecid (5 mM) was added in equal volume to the 96-well plate containing 5 × 10⁴ HEK-293 (AddexBio, San Diego, CA) cells/well. The plates were allowed to equilibrate for 1 h at 37°C with 5% CO_2, and fluorescence (pre-stimulation) was immediately measured in a microplate reader. The cells were then treated with Ar1-Ar24 (16 μg/mL), Ca²⁺ channel inhibitor verapamil (32 μg/mL), and DMSO (mock) at 30 s. Then at 60 s, carbachol (50 μg/mL; calcium channel stimulator) was added, and the fluorescence (post-stimulation) was recorded for an additional 120 s.