COUPLING OF CALCIUM TO THE INTERACTION OF TROPONIN-I WITH TROPONIN-C FROM CARDIAC-MUSCLE

The interaction of troponin I (CTnI) with troponin C (CTnC) from bovine cardiac muscle was studied using CTnC modified at Cys35 and Cys84 with the fluorescent probe 2-[(4'-iodoacetamido)anilino] naphthalene-6-sulfonic acid (CTnC(IAANS)) The association constant for complex formation between the two proteins was determined at 20 degrees C in 0.4 M KCl, 1 mM DTT, 1 mM EGTA, and 25 mM MOPS, pH 7.2. In the presence of EGTA, Mg2+, and Ca2+ these constants were 1.46 X 10(7), 4.1 X 10(7), and 12.7 X 10(7) M(-1), respectively, with corresponding free energy values of -9.62, -10.23, and -10.88 kcal mol(-1). The CTnI.CTnC(IAANs) complex was stabilized by -0.61 kcal when the two Ca/Mg sites of CTnC(IAANS) Were saturated with Mg2+ and by -1.26 kcal when all three Ca2+ sites were occupied by Ca2+. These results suggest that calcium activation in cardiac muscle may be accompanied by a coupling free energy of -0.65 kcal. This value is a factor of 4 smaller than the value previously determined, using a similar method, for the (troponin I).(troponin C) complex from skeletal muscle [Wang, C.-K., & Cheung, H. C. (1985) Biophys. J. 48, 727-739]. Since CTnC has only one Ca2+-specific site and troponin C from skeletal muscle has two such sites, the present result is a factor of 2 smaller than that for the skeletal complex on the basis of a single specific site. Phosphorylation of CTnI by 3',5'-cyclic AMP-dependent protein kinase resulted in a decrease of the association constants by a factor of 2.5-3.5. This decrease is consistent with the known loss of calcium sensitivity induced by phosphorylation of CTnI in cardiac muscle. The lost affinity for CTnC was recovered upon treatment of phosphorylated CTnI with a phosphatase. There was no loss in free energy coupling by calcium in the phosphorylated complex. If the observed free energy coupling reflects the extent of coupling that occurs during activation of the myocardium, phosphorylation of CTnI does not seem to result in impaired Ca2+ activation.