Effects of thin and thick filament proteins on calcium binding and exchange with cardiac troponin C

Understanding the effects of thin and thick. lament proteins on the kinetics of Ca2+ exchange with cardiac troponin C is essential to elucidating the Ca2+-dependent mechanisms controlling cardiac muscle contraction and relaxation. Unlike labeling of the endogenous Cys-84, labeling of cardiac troponin C at a novel engineered Cys-53 with 2-(4'-iodoacetamidoanilo)napthalene-6-sulfonic-acid allowed us to accurately measure the rate of calcium dissociation from the regulatory domain of troponin C upon incorporation into the troponin complex. Neither tropomyosin nor actin alone affected the Ca2+ binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca2+ sensitivity (similar to 7.4-fold) and accelerated the rate of Ca2+ dissociation from the regulatory domain of troponin C (similar to 2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin. laments (actin-tropomyosin-troponin) increased the Ca2+ sensitivity (similar to 6.2-fold) and decreased the rate of Ca2+ dissociation from the regulatory domain of troponin C (similar to 8.1-fold), which was completely reversed by ATP. Consistent with physiological data, replacement of cardiac troponin I with slow skeletal troponin I led to higher Ca2+ sensitivities and slower Ca2+ dissociation rates from troponin C in all the systems studied. Thus, both thin and thick. lament proteins influence the ability of cardiac troponin C to sense and respond to Ca2+. These results imply that both cross-bridge kinetics and Ca2+ dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.