Interactions at the NH2-Terminal Interface of Cardiac Troponin I Modulate Myofilament Activation

Received 23 October 1998; accepted in revised form 23 October 1998Cardiac troponin I (cTnI) is an essential element in activation of myofilaments by Ca2+binding to cardiac troponin C (cTnC). Yet, its role in transduction of the Ca2+binding signal to cardiac troponin T (cTnT) and tropomyosin-actin remain poorly understood. We have recently discovered that regions of cTnI C-terminal to a previously defined inhibitory peptide are essential for full inhibitory activity and Ca2+-sensitivity of cardiac myofilaments (Raricket al1997). 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 residues) and two N-terminal deletion mutants of mouse cTnI, cTnI54–211(missing 53 residues), and cTnI80–211(missing 79 residues). The cTnI54–211mutant retained the ability to bind to cTnT, but lost the ability to bind to cTnC, whereas the cTnI80–211mutant lost the ability to bind to cTnT, but bound weakly to cTnC. Both mutants bound to F-actin. In the absence of Ca2+, cTnI54–211was able to inhibit the unregulated MgATPase activity of myofibrils lacking endogenous cTnI-cTnC to the same extent as WT-cTnI, whereas cTnI80–211had some impairment of its inhibitory capability. Reconstitution with cTnI54–211/cTnC complex did not restore Ca2+-activation of myofibrillar MgATPase activity at all, however, the cTnI80–211/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-binding domain on cTnI. They also indicate that the structural cTnC binding site on cTnI is required for Ca2+-dependent activation of cardiac myofilaments, and that cTnT binding to the N-terminus of cTnI is a negative regulator of activation.