Excitation-contraction coupling events leading to the onset of contraction were studied in single skeletal frog muscle fibers. This entailed the simultaneous measurement of the changes in intracellular calcium concentration using antipyrylazo III and fura-2, isometric force, and clamp voltage in a modified single vaseline gap chamber for the first time. The calcium transients were incorporated into an analysis of calcium binding to regulatory sites of troponin C (TnC) that permitted both a linear and a cooperative interaction. The analysis assumed that the onset of mechanical activation corresponds with a particular TnC saturation with calcium setting constraints for the calcium binding parameters of the regulatory sites. Using a simple model that successfully reproduced both the time course and the relative amplitudes of the measured isometric force transients over a wide membrane potential range, k(off) of TnC was calculated to be 78 s-1 for the cooperative model at 10°C. Together with the above constraints this gave a dissociation constant of 8.8 ± 2.5 μM and a relative TnC saturation at the threshold (S(th)) that would cause just detectable movement of 0.17 ± 0.03 (n = 13: mean ± SE). The predictions were found to be independent of the history of calcium binding to the regulatory sites. The observed delay between reaching S(th) and the onset of fiber movement (8.7 ± 1.0 ms; mean ± SE, n = 37: from seven fibers) was independent of the membrane potential giving an upper estimate for the delay in myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained structural conditions close to those in vivo.
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