Depolarization-Induced Calcium Release from Isolated Triads Measured With Impermeant Fura-2

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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 μ 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.



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