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

• Published in: Biophysical journal Vol. 92; no. 9; pp. 3195 - 3206
• Main Authors: Davis, Jonathan P., Norman, Catalina, Kobayashi, Tomoyoshi, Solaro, R. John, Swartz, Darl R., Tikunova, Svetlana B.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-05-2007; Biophysical Society
• Subjects: Actins - metabolism; Animals; Biophysics; Calcium; Calcium - metabolism; Cells, Cultured; Heart; Kinetics; Male; Muscle and Contractility; Muscular system; Myocardial Contraction - physiology; Myocytes, Cardiac - metabolism; Myosins - metabolism; Protein Binding; Proteins; Rats; Sarcomeres - physiology; Troponin C - metabolism
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1529/biophysj.106.095406

Understanding the effects of thin and thick filament proteins on the kinetics of Ca
2+ exchange with cardiac troponin C is essential to elucidating the Ca
2+-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 Ca
2+ binding properties of the troponin complex. However, addition of actin-tropomyosin to the troponin complex decreased the Ca
2+ sensitivity (∼7.4-fold) and accelerated the rate of Ca
2+ 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 Ca
2+ sensitivity (∼6.2-fold) and decreased the rate of Ca
2+ 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 Ca
2+ sensitivities and slower Ca
2+ 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 Ca
2+. These results imply that both cross-bridge kinetics and Ca
2+ dissociation from troponin C work together to modulate the rate of cardiac muscle relaxation.