Monitoring the Structural Behavior of Troponin and Myoplasmic Free Ca Concentration during Twitch of Frog Skeletal Muscle

The interaction of troponin molecules on the thin filament with Ca²⁺ plays a key role in regulating muscle contraction. To characterize the structural changes of troponin caused by Ca²⁺ and crossbridge formation, we recorded the small-angle x-ray intensity and the myoplasmic free Ca²⁺ concentration using fluo-3 AM in the same frog skeletal muscle during twitch elicited by a single electrical pulse at 16°C. In an overstretched muscle, the intensity of the meridional reflection from troponin at 1/38.5 nm⁻¹ began to change at 4 ms after the stimulus, reached a peak at 10 ms, and returned to the resting level with a halftime of 25 ms. The concentration of troponin-bound Ca²⁺ began to increase at 1-2 ms after the stimulus, reached a peak at 5 ms, and returned to the resting level with a halftime of 40 ms, indicating that troponin begins to change conformation only after a sizable amount of Ca²⁺ has bound to it, and returns to the resting structure even when there is still some bound Ca²⁺. In a muscle with a filament overlap, crossbridge formation appears to slow down Ca²⁺ release from troponin and have a large effect on its conformation.