Rapid filtration measurements of Ca2+ release from cisternal sarcoplasmic reticulum vesicles

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Abstract
A radioactive tracer and rapid filtration method was applied to the study of Ca2+ release from sarcoplasmic reticulum (SR) vesicles which were preloaded passively (equilibration with millimolar Ca2+) or actively (in the presence of ATP or acetyl phosphate). The method allows complete substitution of the loading mixture with release medium in constant flow, and time resolution between 0.01 and 10.0 s. Net release can be clearly distinguished from isotope exchange. The latter is prominent in longitudinal SR vesicles. Net Ca2+ release is observed only from cisternal SR vesicles, is Ca2+ (micromolar) dependent, and is accelerated by inactive ATP analogues, or ATP itself, even in the presence of Mg2+. Net release has a strong pH dependence (between 6 and 7), and very little temperature dependence (consistent with a passive channel). In media of physiological significance (1 mM ATP, 1 mM magnesium, and free Ca2+ in the micromolar range), net Ca2+ release proceeds with a rate constant of approx. 100 s−1.