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

Summary 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 m 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 Ca²⁺-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.We thank Gerry Vaio and Melanie Vander Klok for excellent technical support. This work was supported by the National Institutes of Health program project grant PO1-HL27867, NSF Biological Instrumentation Grant DIR-8812094 and State of Ohio Research Challenge Grant.