Recordings of ER and cytoplasmic Ca2+ in HEK293 cells
HEK293 cells, stably and inducible-expressing WT and mutant R2474S RyR2, were generated as described and provided by Dr. Takashi Murayama (Juntendo University). Cells co-express R-CEPIA1er, a calcium-measuring organelle-entrapped protein indicator located at the ER, and they have an inducible expression for wild-type RyR2 and mutant RyR2 R2473S isoforms. 60.000 cells/well were seeded onto 96-well plates coated with 50 µg/ml poly-D-Lysine, as described previously. Cells were grown in 10% FBS, DMEM media, at 37ºC and 5% CO2 for 24 hours. On day 2, RyR expression was induced with 2 µg/ml doxycycline. Induction was achieved after 24-28 hours, when calcium measurements were performed in HEPES-buffered Krebs solution (140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 11 mM D-(+)-Glucose, 5 mM HEPES, distilled H2O to 450 mL, pH 7.4). Fluorescent ratios (F/F0) were calculated as the ratio of the fluorescence intensities between the initial (F0) and the last (F) 100 seconds by R-CEPIA1er in the Glomax Discover Microplate Reader (Promega). Caffeine-induced Ca2+ transients were also measured with Fura2 AM at room temperature using an ECLIPSE Ti/L100 epifluorescence microscope (Nikon) equipped with a 20X S-Fluor objective, a lambda-DG4 illumination system, an Orca-Flash 2.8 camera (Hamamatsu) and NisElements-AR software.