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 Ca²⁺ concentration. Tn is composed of TnC, TnI, and TnT. Ca²⁺ 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 Ca²⁺ 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 Ca²⁺, 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 Ca²⁺ 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 Ca²⁺, there was little difference in the activation of ATPase activity between these preparations.Striated muscle thin filaments exist in equilibrium among multiple states. Ca²⁺ binding to the regulatory domain of troponin C (TnC)² along the thin filaments and strong cross-bridge interactions with thick filaments are thought to shift the equilibrium. Ca²⁺ binds to the regulatory domain of TnC, which regulates the interaction of troponin I (TnI) with actin-tropomyosin (Tm) and TnC (1–3). In the thin filaments, the inhibitory region of TnI (residues 104–115 of rabbit fast skeletal TnI (fsTnI) or 138–149 of mouse cardiac TnI (cTnI)) undergoes a structural transition depending on the Ca²⁺ state of TnC (4, 5). In the absence of Ca²⁺ at the regulatory site(s) of TnC, the inhibitory region interacts with actin to prevent activation of myosin ATPase activity. When Ca²⁺ binds to the regulatory site(s) of TnC, the switch region of TnI, which is located at the C terminus of the inhibitory region, interacts with the newly exposed hydrophobic patch of the N-terminal regulatory domain of TnC (6–8). This interaction causes the removal of the inhibitory region and the second actin-Tm binding region of TnI from the actin surface and allows actin to interact with myosin. In the presence of Ca²⁺ at the regulatory sites of TnC, the inhibitory region and the central helical region of TnC are mutually stabilized, according to the recent x-ray crystal structure of the core domain of the fsTn complex (9). The sequence variations in the N-terminal and the C-terminal regions of TnT, another component of the Tn complex, are known to alter the Ca²⁺ sensitivity of myofilament activity (10, 11). In addition, TnT is involved in the Ca²⁺-dependent interaction of the Tn complex with actin-Tm (12). However, the molecular mechanism whereby TnT participates in the Ca²⁺ regulation has not been established.There is evidence supporting the idea that each amino acid residue in the inhibitory region of TnI contributes differently and to a different degree to myofilament activities. One example is genetic mutations and phosphorylation of amino acid residues in the inhibitory region of cardiac TnI that cause the modification of myofilament activities. In hypertrophic or restrictive cardiomyopathy-linked mutations found in the inhibitory region, such as R142Q, L145Q, and R146G/Q/W mutations (mouse cTnI sequence number), induce Ca²⁺ sensitization of myofilament activities and an increase in ATPase/tension at low Ca²⁺] (13, 14). Recently we reported that thin filaments reconstituted with R146G or R146W mutant cTnI bind Ca²⁺ tighter than those with cTnI(wt) (15). The Ca²⁺ sensitization may occur as a result of the destabilization of the off-state of the thin filaments due to the mutation introduced into the actin-Tm-interacting residue, i.e. Arg-146, of cTnI. On the other hand, Thr-144 is phosphorylated by protein kinase C (PKC) specifically, although the consequence of the PKC-dependent phosphorylation of Thr-144 has not yet been clearly defined. Pseudophosphorylation of Thr-144 was shown to cause Ca²⁺ desensitization in in vitro motility assays (16), whereas there is a report that indicates phosphorylation of Thr-144 sensitizes skinned cardiomyocytes to Ca²⁺ (17). Furthermore, Tachampa et al. reported that Thr-144 of cTnI is important for length-dependent activation of skinned cardiac muscle (18). Thus in each case presented above, a specific change in a single amino acid in the inhibitory region of TnI induced different and divergent effects on myofilament activities.Our aim of this study is to assess the functional contributions of the individual amino acid residues in the inhibitory region to the regulatory function. To assess the functional roles of the individual amino acid residues systematically, we used Ala scanning (19, 20). Ala substitution deletes all the interactions made by atoms beyond β-C yet does not alter the peptide backbone conformation, unless it is applied to Gly or Pro. Ala is one of the most abundant amino acids and is found in both buried and exposed positions. We found that almost the entire minimum inhibitory region of cTnI we investigated (Fig. 1) is important for both the inhibition and activation. Our data also indicate that the C-terminal part of the inhibitory region destabilizes the active state of the thin filaments. We also found that Thr-144 is involved in NEM-S1-dependent activation of ATPase activity in the absence of Ca²⁺.Open in a separate windowFIGURE 1.Inhibitory region of TnI. A, sequence comparison of the minimum inhibitory region from various vertebrates. The amino acid residues that are different from fsTnI are colored green in cardiac sequences. Note the amino acid sequence of the inhibitory region is highly conserved. Also the amino acid sequences of the minimum inhibitory region of the mutants we investigated in this study are shown. B, crystal structure of the inhibitory region and its surrounding region in chicken fsTn complex in the Ca²⁺-bound form (PDB: 1YTZ). TnC, pink; TnT, light blue; TnI, gray. The segment, corresponding to residues 143–149 of mouse cTnI, is colored red.