Cell culture and transfection of HEK293 cells

HEK293 cells were cultured in Earle's Minimum Essential Medium (Sigma, Munich, Germany), supplemented with 10% fetal calf serum (Gibco Thermo Fisher Scientific, Darmstadt, Germany), 2 mM l‐glutamine, 100 U·mL⁻¹ penicillin and 0.1 mg·mL⁻¹ streptomycin. To characterize human TRPC6 mutants and to verify the efficiency of larixol derivatives, parental HEK293 cells were seeded on 25 mm glass coverslips and transfected with 4 μL Fugene HD (Promega, Mannheim, Germany) and 2 μg of DNA encoding wild‐type or mutated variants of TRPC6‐YFP. For FRET assays, HEK293 cells were co‐transfected with 0.5–0.8 μg pcDNA3‐hTRPC6‐cyan fluorescent protein (CFP) wild‐type and 1.2–1.5 μg pcDNA3‐hTRPC6‐YFP mutant to achieve a 1:1 molar ratio between YFP‐fused and CFP‐fused channel constructs. After 24 h, cells were directly used for intracellular Ca²⁺ concentration ([Ca²⁺]_i) imaging and FRET measurements, or singularized with 0.25% trypsin for electrophysiological experiments. Fluorescence plate imaging analyses of cells expressing TRPC6 mutants were realized with stably transfected polyclonal cell lines, selected by adding 1 mg·mL⁻¹ geneticin (G418) to the medium. All cells were maintained at 37°C and in a 5% CO₂‐aerated, humidified atmosphere.