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

Abstract

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 (R(max)). Steady- state [Ca](SR)/[Ca](c) was ~7000. Therefore the resting [Ca](SR) may approach 700 μM in the rat cardiac myocyte at [Ca](c) = 100 nM. The sarcoplasmic reticulum (SR) Ca pump requires a free energy of ΔG ≃ 44 kJ · mol-1 to generate this [Ca] gradient (e.g., ~74% of ΔG(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 ~400 μmol/liter cytosolic volume. Used together, these data allowed evaluation of the in situ SR Ca-buffering properties. The SR Ca-binding site concentration was ~14 mM, and K(d(Ca)) ≃ 0.638 mM [Ca](SR).