Measuring Sarcoplasmic Reticulum Calcium Uptake

SR calcium uptake was measured using a previously described Millipore filtration technique.²⁰ Approximately 150 μg of ventricular protein extract was incubated at 37 °C in 1.5 mL of calcium uptake medium (40 mmol/L imidazole, pH 7.0, 100 mmol/L KCl, 5 mmol/L MgCl₂, 5 mmol/L NaN₃, 5 mmol/L potassium oxalate, 0.5 mmol/L EGTA), and various concentrations of CaCl₂ to yield 0.03–3 μmol/L free calcium. Ruthenium red was added to a final concentration of 1 μmole immediately before adding substrates to begin calcium uptake. A final concentration of 5 mmole ATP was added to initiate the reaction. The reaction was terminated at 1 minute by filtration. The rate of SR calcium uptake and the calcium concentration required for EC50 were determined by nonlinear curve fitting analysis using GraphPad Prism v6.01 software (GraphPad, San Diego, CA).