Ala scanning of the inhibitory region of cardiac troponin I.

In skeletal and cardiac muscles, troponin (Tn), which resides on the thin filament, senses a change in intracellular Ca(2+) concentration. Tn is composed of TnC, TnI, and TnT. Ca(2+) binding to the regulatory domain of TnC removes the inhibitory effect by TnI on the contraction. The inhibitory region of cardiac TnI spans from residue 138 to 149. Upon Ca(2+) activation, the inhibitory region is believed to be released from actin, thus triggering actin-activation of myosin ATPase. In this study, we created a series of Ala-substitution mutants of cTnI to delineate the functional contribution of each amino acid in the inhibitory region to myofilament regulation. We found that most of the point mutations in the inhibitory region reduced the ATPase activity in the presence of Ca(2+), which suggests the same region also acts as an activator of the ATPase. The thin filaments can also be activated by strong myosin head (S1)-actin interactions. The binding of N-ethylmaleimide-treated myosin subfragment 1 (NEM-S1) to actin filaments mimics such strong interactions. Interestingly, in the absence of Ca(2+) NEM-S1-induced activation of S1 ATPase was significantly less with the thin filaments containing TnI(T144A) than that with the wild-type TnI. However, in the presence of Ca(2+), there was little difference in the activation of ATPase activity between these preparations.