Effects of Thin and Thick Filament Proteins on Calcium Binding and Exchange with Cardiac Troponin C

Understanding the effects of thin and thick filament 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 (∼7.4-fold) and accelerated the rate of Ca2+ dissociation from the regulatory domain of troponin C (∼2.5-fold). Subsequent addition of myosin S1 to the reconstituted thin filaments (actin-tropomyosin-troponin) increased the Ca2+ sensitivity (∼6.2-fold) and decreased the rate of Ca2+ dissociation from the regulatory domain of troponin C (∼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 filament 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.