Interactions at the NH2-terminal interface of cardiac troponin I modulate myofilament activation

Cardiac troponin I (cTnI) is an essential element in activation of myofilam ents by Ca2+ binding to cardiac troponin C (cTnC). Yet, its role in transdu ction of the Ca2+ binding signal to cardiac troponin T (cTnT) and tropomyos in-actin remain poorly understood. We have recently discovered that regions of cTnI C-terminal to a previously defined inhibitory peptide are essentia l for full inhibitory activity and Ca2+-sensitivity of cardiac myofilaments (Rarick et al., 1997). However, apart from its role in structural binding to cTnC. there is little knowledge concerning the role of the N-terminus of cTnI in the activation and regulation of cardiac myofilaments. To address this question, we generated wild-type mouse cardiac TnI (WT-cTnI: 211 resid ues) and two N-terminal deletion mutants of mouse cTnI, cTnI(54-211) (missi ng 53 residues), and cTnI(80-211) (missing 79 residues). The cTnI(54-211) m utant retained the ability to bind to cTnT, but lost the ability to bind to cTnC, whereas the cTnI(80-211) mutant lost the ability to bind to cTnT, bu t bound weakly to cTnC. Both mutants bound to F-actin. In the absence of Ca 2+. cTnI(54-211) was able to inhibit the unregulated MgATPase activity of m yofibrils lacking endogenous cTnI-cTnC to the same extent as WT-cTnI, where as cTnI(80-211) had some impairment of its inhibitory capability. Reconstit ution with cTnI(54-211)/cTnC complex did not restore Ca2+-activation of myo fibrillar MgATPase activity at all. however, the cTnI(80-211)/cTnC complex restored Ca2+-activation to nearly 50% of that obtained with WT-cTnI/cTnC. These data provide the first evidence of a significant function of a cTnT-b inding domain on cTnI. They also indicate that the structural cTnC binding site on cTnI is required for Ca2+-dependent activation of cardiac myofilame nts, and that cTnT binding to the N-terminus of cTnI is a negative regulato r of activation. (C) 1999 Academic Press.