Ca2+ Transport Assay of SERCA2a

Ca²⁺-transport assays were performed with similar porcine SR samples as in the Ca²⁺-ATPase assays described above. The compound effect on the Ca²⁺-transport activity of SERCA2a was determined using an oxalate-supported assay in which the change in fluorescence in a Ca-sensitive dye, Fluor-4, was determined as previously described^{18 (link)}. A buffered solution containing 50 mM MOPS (pH 7.0), 100 mM KCl, 30 mg/mL sucrose, 1 mM EGTA, 10 mM potassium oxalate, 2 μM Fluo-4, 30 μg/mL porcine cardiac SR vesicles, CaCl₂ calculated to reach the free [Ca²⁺] (pCa 8.0, 6.2, and 5.4), and compound (0.048 to 50μM) was dispensed into 384-well black walled, transparent bottomed plates (Greiner Bio-One) containing the tested small molecule and incubated at 22°C for 20 minutes while covered and protected from light. To start the reaction, MgATP was added to a final concentration of 5 mM, and the decrease in 485-nm excited fluorescence of Fluo-4 was monitored at 520 nm for 15 min using a FLIPR Tetra (Molecular Devices, San Jose, CA).