Quantitative Measurement of Ca2+ in the Sarcoplasmic Reticulum Lumen of Mammalian Skeletal Muscle

Skeletal muscle stores Ca2+ in the sarcoplasmic reticulum (SR) and releases it to initiate contraction, but the concentration of luminal Ca2+ in the SR ([Ca2+](SR)) and the amount that is released by physiological or pharmacological stimulation has been difficult to measure. Here we present a novel, yet simple and direct, method that provides the first quantitative estimates of static content and dynamic changes in [Ca2+](SR) in mammalian skeletal muscle, to our knowledge. The method uses fluo-5N loaded into the SR of single, mammalian skeletal muscle cells (murine flexor digitorum brevis myofibers) and confocal imaging to detect and calibrate the signals. Using this method, we have determined that [Ca2+](SR,) (free) is 390 mu M. 4-Chloro-m-cresol, an activator of the skeletal muscle ryanodine receptor, reduces [Ca2+](SR, free) to similar to 8 mu M, when values are corrected for background fluorescence from cytoplasmic pools of dye. Prolonged electrical stimulation (10 s) at 50 Hz releases 88% of the SR Ca2+ content, whereas stimulation at 1 Hz (10 s) releases only 20%. Our results lay the foundation for molecular modeling of the dynamics of luminal SR Ca2+ and for future studies of the role of SR Ca2+ in healthy and diseased mammalian muscle.