Abstract
Call efflux from frog muscle sarcoplasmic reticulum (SR) vesicles was studied by measuring external free [Ca 2+] using Fluo-3 fluorescence. Light SR vesicles were preloaded with Ca 2+ in the presence of ATP and inorganic phosphate (P i). Calcium pump reversal was activated by either depletion of the medium ATP by apyrase in the presence of 20 mM P i, or resuspending preloaded vesicles in an ATP-free solution containing 1 mM ADP and 20 mM P i. Cyclopiazonic acid (CPA) and thapsigargin (TG), at concentrations of 2.5 μM, which completely inhibit Ca 2+ uptake, both inhibited the pump reversal efflux almost completely. When active Ca 2+ uptake was stopped by either ATP-depletion or addition of CPA, a leak efflux of 6–7 nmole/mg/min was recorded. TG (2.5 μM) reduced this leak by over 50%, suggesting that TG, but not CPA, can slow the passage of calcium ions through the Ca 2+-ATPase passive channel.
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