Calcium Uptake Assay in Membrane Vesicles

Membrane vesicles were resuspended in 1 mL buffer (30 mmol/L Tris-HCl, pH 7.0; 100 mmol/L KCl; 5 mmol/L NaN₃; 5 mmol/L MgCl₂; 0.15 mmol/L EGTA; 0.12 mmol/L CaCl₂; 1 μCi ⁴⁵Ca²⁺; and 10 mmol/L potassium oxalate) and were divided into 2 × 500-μL aliquots. One aliquot was incubated with a SERCA2a inhibitor cocktail (10 μmol/L thapsigargin, 100 μmol/L ammonium molybdate, and 0.05 μg bafilomycin) and the other remained untreated. Ca²⁺ uptake assays were conducted as described by Andersen (30 (link),31 (link)) using Na₂ATP to initiate uptake. In assays involving rat heart membrane vesicles, 50 μmol/L ryanodine was added to the incubation media for 10 min to inhibit or close the ryanodine receptor Ca²⁺ release channel.