Basic residues within the cardiac troponin T C terminus are required for full inhibition of muscle contraction and limit activation by calcium

Abstract

Striated muscle is activated by myosin- and actin-linked processes, with the latter being regulated through changes in the position of tropomyosin relative to the actin surface. The C-terminal region of cardiac troponin T (TnT), a tropomyosin-associated protein, is required for full TnT inactivation at low Ca2+ and for limiting its activation at saturating Ca2+. Here, we investigated whether basic residues in this TnT region are involved in these activities, whether the TnT C terminus undergoes Ca2+- dependent conformational changes, and whether these residues affect cardiac muscle contraction. We generated a human cardiac TnT variant in which we replaced seven C-terminal Lys and Arg residues with Ala and added a Cys residue at either position 289 or 275 to affix a fluorescent probe. At PCA 3.7, actin filaments containing high-alanine TnT had an elevated ATPase rate like that obtained when the last TnT 14 residues were deleted. Acrylodan-tropomyosin fluorescence changes and S1-actin binding kinetics revealed that at PCA 8, the high-alanine TnT-containing filaments did not enter the first inactive state. FRET analyses indicated that the C-terminal TnT region approached Cys-190 of tropomyosin as actin filaments transitioned to the inactive B state; that transition was abolished with high-alanine TnT. High-alanine TnT-containing cardiac muscle preparations had increased Ca2+ sensitivity of both steadystate isometric force and sinusoidal stiffness as well as increased maximum steady-state isometric force and sinusoidal stiffness. We conclude that C-terminal basic residues in cardiac TnT are critical for the regulation of cardiac muscle contraction.