Assessment of intra-SR free [Ca] and buffering in rat heart

To measure the free intrasarcoplasmic reticulum [Ca] ([Ca]SR) in isolated rat cardiac microsomes, ventricular tissue was homogenized in the presence of the low-affinity Ca indicator furaptra. Stepwise increases in cuvette [Ca] ([Ca]c) in the presence of ATP caused progressive increases in steady-state intravesicular fluorescence ratio to a maximum (Rmax). Steady-state [Ca]SR/[Ca]c was approximately 7000. Therefore the resting [Ca]SR may approach 700 microM in the rat cardiac myocyte at [Ca]c = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of deltaG approximately 44 kJ x mol(-1) to generate this [Ca] gradient (e.g., approximately 74% of deltaG(ATP)). Total SR 45Ca uptake was also measured in digitonin-permeabilized myocytes as a function of [Ca]c in the absence of precipitating ions. The steady-state SR Ca content at 100 nM [Ca]c was approximately 400 micromol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was approximately 14 mM, and Kd(Ca) approximately 0.638 mM [Ca]SR