Depolarization-induced calcium release from isolated triads measured with impermeant fura-2

Depolarization-induced Ca2+ release was studied in a mixture of triads and terminal cisternae isolated from rabbit skeletal muscle. The vesicles were actively loaded with known amounts of Ca2+ in the absence of precipitating anions in a solution containing 100 mM K propionate buffer. Changes in extravesicular Ca2+ were monitored with 10 microM Fura-2 (membrane impermeant form). Ca2+ release was initiated by diluting an aliquot of the loaded vesicles into a TEACl release solution designed to maintain a constant [K+].[Cl-] product. Fast release, defined as the percentage of total Ca2+ loaded which released in less than 10 sec, occurred when extravesicular free Ca2+ was in the submicromolar range and was unaffected by 5 mM caffeine under depolarizing conditions, change in external pH to 6.5, and an increase in external Mg2+ concentration from 0.1 to 0.2 mM. Thus, the Ca2+ release measured in these studies is distinct from Ca(2+)-induced Ca2+ release. The fast release more than doubled when a greater dilution (1:20 versus 1:10) of the loaded vesicles into the release solution, which would produce a larger depolarization, was used. The percentage of loaded Ca2+ which released rapidly in a particular triad preparation was similar to the percentage of vesicles structurally coupled as visualized by electron microscopy.