KINETICS OF CONTRACTILE ACTIVATION IN VOLTAGE-CLAMPED FROG SKELETAL-MUSCLE FIBERS

Excitation-contraction coupling events leading to the onset of contrac tion 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 cal cium binding to regulatory sites of troponin C (TnC) that permitted bo th a linear and a cooperative interaction. The analysis assumed that t he onset of mechanical activation corresponds with a particular TnC sa turation with calcium setting constraints for the calcium binding para meters of the regulatory sites, Using a simple model that successfully reproduced both the time course and the relative amplitudes of the me asured isometric force transients over a wide membrane potential range , k(off) of TnC was calculated to be 78 s(-1) for the cooperative mode l at 10 degrees C, Together with the above constraints this gave a dis sociation constant of 8.8 +/- 2.5 mu M and a relative TnC saturation a t 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 in dependent of the history of calcium binding to the regulatory sites. T he observed delay between reaching S,, and the onset of fiber movement (8.7 +/- 1.0 ms; mean +/- SE, n = 37; from seven fibers) was independ ent of the membrane potential giving an upper estimate for the delay i n myofilament activation. We thus emerge with quantitative values for the calcium binding to the regulatory sites on TnC under maintained st ructural conditions close to those in vivo.