Cell cultures and transfection

Experiments were performed on human embryonic kidney 293 cells (HEK293; American Type Culture Collection), which were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 50 U/mL penicillin, and 50 μg/mL streptomycin in a humidified 5% CO₂ atmosphere at 37°C. Cells were cultured in 75-cm² plastic culture flasks (NUNC, Rochester, NY, USA) for 36–72 h until they reached 80–95% confluence. Before the day of transfection, ~150 000 cells were plated on 35 mm culture dishes (Sarstedt, Newton, NC, USA) and incubated at 37°C for at least 24 h. For each culture dish of HEK293 cells, transfection of wild-type P2X4R was conducted using 2 μg of DNA with 2 μl of jetPRIME reagent in 2 ml of Dulbecco modified Eagle’s medium, according to the manufacturer’s instructions (PolyPlus-transfection, Illkirch, France). After 24–48 h of incubation, the transfected cells were mechanically dispersed and re-cultured on 35 mm dishes of Corning 3294 CellBIND Surface for 1–4 hours before recording. Transfected cells were identified by the fluorescence signal of enhanced green fluorescent protein using an inverted research microscope with fluorescence illuminators (Model IX71; Olympus, Melville, NY).