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

Thomas R. Shannon, Donald M Bers

Research output: Contribution to journalArticle

86 Scopus citations

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).

Original languageEnglish (US)
Pages (from-to)1524-1531
Number of pages8
JournalBiophysical Journal
Volume73
Issue number3
StatePublished - Sep 1997
Externally publishedYes

ASJC Scopus subject areas

  • Biophysics

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