Ala Scanning of the Inhibitory Region of Cardiac Troponin I

Abstract

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.Striated muscle thin filaments exist in equilibrium among multiple states. Ca 2ϩ binding to the regulatory domain of troponin C (TnC) 2 along the thin filaments and strong crossbridge interactions with thick filaments are thought to shift the equilibrium. Ca 2ϩ binds to the regulatory domain of TnC, which regulates the interaction of troponin I (TnI) with actintropomyosin (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 Ca 2ϩ state of TnC (4, 5). In the absence of Ca 2ϩ at the regulatory site(s) of TnC, the inhibitory region interacts with actin to prevent activation of myosin ATPase activity. When Ca 2ϩ 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 . 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 Ca 2ϩ 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 Ca 2ϩ sensitivity of myofilament activity (10, 11). In addition, TnT is involved in the Ca 2ϩ -dependent interaction of the Tn complex with actin-...

Phosphorylation of Cardiac Troponin I at Protein Kinase C Site Threonine 144 Depresses Cooperative Activation of Thin Filaments

“…Every 2 min (up to 12 min), a 10-l aliquot was removed into 90 l of 0.2 M perchloric acid to terminate the reaction. ATPase activity was determined from the time course (typically 6 time points/one time course) of phosphate liberation using the malachite green method (14,29). In the NEM-S1 ATPase activity measurements, the initial concentrations of S1, actin, tropomyosin, troponin were 0.2, 5.0, 1.0, 1.0 M with concentrations increased by an amount equal to the NEM-S1 to maintain the same level of free actin/tropomyosin/ troponin (6,14).…”

“…Thr-144 is unique to cardiac TnI (slow and fast skeletal TnI have Pro at the corresponding position). It has been demonstrated that Thr-144 is involved in length-dependent activation of cardiac muscle fibers (13) and strong cross-bridgedependent activation of actomyosin S1-ATPase activity (14).…”

“…To assess the effects of strong cross-bridge-dependent activation, we measured actomyosin S1-ATPase activity in the presence of NEM-treated S1 (NEM-S1). NEM-S1 mimics the strong cross-bridge, binds strongly to actin even in the presence of ATP, and activates the thin filaments to fully active state (6,14,39). Fig.…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…Thr143 is a cardiac-specific residue (proline in skeletal muscle) located in the inhibitory region of cTnI (residues 137-148 of the human cTnI sequence). In the absence of Ca 2+ , this region interacts with actin and thereby inhibits actomyosin ATPase activity, while upon Ca 2+ binding to cTnC, the inhibitory region interacts with the N-terminal regulatory domain of cTnC, allowing actin to interact with myosin Kobayashi et al 2009;McKay et al 1999). Alanine substitution of Thr143 did not have any significant effect on the Ca 2+ -sensitivity of force, but there was the involvement of strong cross-bridge-dependent activation of the thin filaments in the absence of Ca 2+ (Table 1) (Kobayashi et al 2009).…”

“…In the absence of Ca 2+ , this region interacts with actin and thereby inhibits actomyosin ATPase activity, while upon Ca 2+ binding to cTnC, the inhibitory region interacts with the N-terminal regulatory domain of cTnC, allowing actin to interact with myosin Kobayashi et al 2009;McKay et al 1999). Alanine substitution of Thr143 did not have any significant effect on the Ca 2+ -sensitivity of force, but there was the involvement of strong cross-bridge-dependent activation of the thin filaments in the absence of Ca 2+ (Table 1) (Kobayashi et al 2009). Also, a reduction in myofilament length-dependent activation has been observed in cardiac rat trabeculae expressing slow skeletal troponin (ssTn), which has a proline at position 122 versus a threonine at position 143 in cTnI (Table 1) (Tachampa et al 2007).…”