AbstractActivation of striated muscle contraction occurs in response to Ca2+ binding to troponin C (TnC). The resulting reorganization of troponin repositions tropomyosin on actin and permits activation of myosin catalyzed ATP hydrolysis. It now appears that the levels of activity at both low and saturating Ca2+ are modulated by the C-terminal 14 amino acids of cardiac troponin T (TnT). We made a series of mutants of human cardiac troponin T, isoform 2, with deletions from the C-terminal end: Δ4, Δ6, Δ8, Δ10 and Δ14. We measured the effect of these mutations on the normalized ATPase activity at saturating Ca2+, the change in acrylodan tropomyosin fluorescence at low Ca2+, and the degree of Ca2+ stimulation of the rate of binding of rigor myosin S1 to pyrene-labeled actin-tropomyosin-troponin. Together, these measurements define the distribution of actin-tropomyosin-troponin among the 3 regulatory states. Results from rates of rigor S1 binding deviated from other measurements when > 8 residues of TnT were deleted. That deviation was due to increased rates of binding of rigor S1 to pyrene-labeled actin with truncated TnT at saturating Ca2+. Such behavior violated a key assumption in the determination of the B state by this method. Nevertheless, all methods show that as residues were removed from the C-terminus of TnT there was approximately a proportional loss of the inactive B state at low Ca2+ and an increase in the active M state at saturating Ca2+. Most of the C-terminal 14 residues of human cardiac troponin T are essential for forming the inactive B state at low Ca2+ and for limiting the formation of the active M state at saturating Ca2+.
Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium
