Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Lopez-Davila, Alfredo J.; Elhamine, Fatiha; Rueß, Daniel; Papadopoulos, Simon; Iorga, Bogdan; Kulozik, F.P.; Zittrich, Stefan; Solzin, Johannes; Pfitzer, Gabriele; Stehle, Robert

doi:10.1016/j.bpj.2012.08.022

Cited by 5 publications

(14 citation statements)

References 40 publications

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“…1,100 s −1 , is consistent with previous estimates of the rate of Ca 2+ binding to the regulatory sites of troponin C from skeletal muscle in isolated TnC (12,13,24), in whole troponin and reconstituted thin filaments (25,26), and in myofibrils (26). The largest component of the change in orientation of the E helix in the C lobe of TnC also has a rate of ca.…”

Section: Discussionsupporting

confidence: 91%

“…Phase 2, with a rate constant of 120 s −1 coincides with the smaller, slower component of signals from probes on troponin observed in some previous studies using isolated proteins (146 s −1 at 25°C) (23) and myofibrils (150 s −1 at 10°C) (26). Phase 2 also has a similar time course as the azimuthal motion of tropomyosin, as reported by the changes in the intensity of the second actin layer line measured in X-ray diffraction studies of activation in intact amphibian muscle (20,29).…”

Section: Discussionsupporting

confidence: 82%

See 1 more Smart Citation

Structural dynamics of troponin during activation of skeletal muscle

Fusi

Brunello

Sevrieva

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Time-resolved changes in the conformation of troponin in the thin filaments of skeletal muscle were followed during activation in situ by photolysis of caged calcium using bifunctional fluorescent probes in the regulatory and the coiled-coil (IT arm) domains of troponin. Three sequential steps in the activation mechanism were identified. The fastest step (1,100 s −1 ) matches the rate of Ca 2+ binding to the regulatory domain but also dominates the motion of the IT arm. The second step (120 s −1 ) coincides with the azimuthal motion of tropomyosin around the thin filament. The third step (15 s −1 ) was shown by three independent approaches to track myosin head binding to the thin filament, but is absent in the regulatory head. The results lead to a four-state structural kinetic model that describes the molecular mechanism of muscle activation in the thin filament-myosin head complex under physiological conditions. muscle regulation | excitation-contraction coupling | muscle signaling C ontraction of skeletal and cardiac muscle is initiated by a transient increase in the concentration of intracellular Ca 2+ ions, which bind to troponin in the thin filaments of the muscle sarcomere. This leads to azimuthal movement of tropomyosin around the thin filament, which uncovers the myosin binding sites on actin and allows the head domain of myosin from the thick filaments to bind to actin and generate force (1, 2). In vitro studies using isolated protein components showed that myosin head binding can produce a further motion of tropomyosin, at least in low [ATP] or rigor-like conditions (2-4), but the functional significance of this effect in physiological conditions and intact sarcomeres is not clear.To elucidate the molecular structural basis of muscle regulation and the role of myosin binding in situ, we introduced bifunctional fluorescent probes into the calcium-binding subunit of troponin, troponin C (TnC) (Fig. 1, yellow), in demembranated fibers from skeletal muscle (5-7). One probe cross-linked a pair of cysteines introduced into the C helix of TnC (Fig. 1, green), close to the regulatory Ca 2+ binding sites (Fig. 1, black spheres) in its N-terminal lobe, and reports the rotation and opening of this lobe on binding Ca 2+ (5). The N-lobe opening is associated with binding of the switch peptide of troponin I (TnI) (Fig. 1, blue) to a hydrophobic pocket on its surface, and this is a key step in the signaling pathway of calcium regulation (8, 9).A second probe was attached to the E helix of TnC (Fig. 1, magenta) in its C-terminal lobe, which contains a pair of divalent cation binding sites (Fig. 1, gray spheres) that can bind Mg 2+ as well as Ca 2+ . The C lobe of TnC is clasped between two long helices of TnI, one of which forms a coiled coil with part of the tropomyosin-binding component of troponin, troponin T (TnT) (Fig. 1, orange). The C lobe of TnC and these long TnI and TnT helices form a well-defined structural domain called the "IT arm" (9, 10). Although the C-lobe E helix of TnC is continuous with the N-lob...

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Section: Discussionsupporting

confidence: 91%

Section: Discussionsupporting

confidence: 82%

Structural dynamics of troponin during activation of skeletal muscle

Fusi

Brunello

Sevrieva

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Thin-filament switching and cross-bridge recruitment are controlled by [Ca 2þ ]; therefore, k ACT is expected to be Ca 2þ dependent, as shown in Fig. 7 A and previously demonstrated for k ACT and k TR (11,26,(47)(48)(49)(50). In contrast, k REL was found to be independent of [Ca 2þ ].…”

Section: Ca 2d Dependence Of Contraction and Relaxation Ratessupporting

confidence: 56%

The Dilated Cardiomyopathy-Causing Mutation ACTC E361G in Cardiac Muscle Myofibrils Specifically Abolishes Modulation of Ca 2+ Regulation by Phosphorylation of Troponin I

Vikhorev

Song

Wilkinson

et al. 2014

Biophysical Journal

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Phosphorylation of troponin I by protein kinase A (PKA) reduces Ca(2+) sensitivity and increases the rate of Ca(2+) release from troponin C and the rate of relaxation in cardiac muscle. In vitro experiments indicate that mutations that cause dilated cardiomyopathy (DCM) uncouple this modulation, but this has not been demonstrated in an intact contractile system. Using a Ca(2+)-jump protocol, we measured the effect of the DCM-causing mutation ACTC E361G on the equilibrium and kinetic parameters of Ca(2+) regulation of contractility in single transgenic mouse heart myofibrils. We used propranolol treatment of mice to reduce the level of troponin I and myosin binding protein C (MyBP-C) phosphorylation in their hearts before isolating the myofibrils. In nontransgenic mouse myofibrils, the Ca(2+) sensitivity of force was increased, the fast relaxation phase rate constant, kREL, was reduced, and the length of the slow linear phase, tLIN, was increased when the troponin I phosphorylation level was reduced from 1.02 to 0.3 molPi/TnI (EC50 P/unP = 1.8 ± 0.2, p < 0.001). Native myofibrils from ACTC E361G transgenic mice had a 2.4-fold higher Ca(2+) sensitivity than nontransgenic mouse myofibrils. Strikingly, the Ca(2+) sensitivity and relaxation parameters of ACTC E361G myofibrils did not depend on the troponin I phosphorylation level (EC50 P/unP = 0.88 ± 0.17, p = 0.39). Nevertheless, modulation of the Ca(2+) sensitivity of ACTC E361G myofibrils by sarcomere length or EMD57033 was indistinguishable from that of nontransgenic myofibrils. Overall, EC50 measured in different conditions varied over a 7-fold range. The time course of relaxation, as defined by tLIN and kREL, was correlated with EC50 but varied by just 2.7- and 3.3-fold, respectively. Our results confirm that troponin I phosphorylation specifically alters the Ca(2+) sensitivity of isometric tension and the time course of relaxation in cardiac muscle myofibrils. Moreover, the DCM-causing mutation ACTC E361G blunts this phosphorylation-dependent response without affecting other parameters of contraction, including length-dependent activation and the response to EMD57033.

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“…2, 3), (Stehle et al 2009;Stehle 2017). Thus, all these force rises primarily reflect cross-bridge turnover kinetics and even the force rise in k ACT -measurements seems to be little further limited by the additional Ca 2+ activation of the thin filament in comparison to k TR -measurements, a common finding in myofibrils and fibres from skeletal and cardiac muscles (Wahr and Rall 1997;Palmer and Kentish 1998;Stehle et al 2002a, b;Tesi et al 2002a, b), and consistent with Ca 2+ rapidly switching on troponin in skeletal and cardiac myofibrils (Solzin et al 2007;Lopez-Davila et al 2012). The observed rate constants of all three type of force rise reflect the sum of apparent rate constants of crossbridge turnover from non-force-generating to force-generating states (f app ) and from force-generating to non-force states via backwards ( f − app ) and forwards (g app ) cycling, respectively.…”

Section: Similarities Of Force Rises Upon [P I ] and [Ca 2+ ] Changesmentioning

confidence: 61%

Kinetic coupling of phosphate release, force generation and rate-limiting steps in the cross-bridge cycle

Stehle

Tesi

2017

J Muscle Res Cell Motil

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A basic goal in muscle research is to understand how the cyclic ATPase activity of cross-bridges is converted into mechanical force. A direct approach to study the chemo-mechanical coupling between P release and the force-generating step is provided by the kinetics of force response induced by a rapid change in [P]. Classical studies on fibres using caged-P discovered that rapid increases in [P] induce fast force decays dependent on final [P] whose kinetics were interpreted to probe a fast force-generating step prior to P release. However, this hypothesis was called into question by studies on skeletal and cardiac myofibrils subjected to P jumps in both directions (increases and decreases in [P]) which revealed that rapid decreases in [P] trigger force rises with slow kinetics, similar to those of calcium-induced force development and mechanically-induced force redevelopment at the same [P]. A possible explanation for this discrepancy came from imaging of individual sarcomeres in cardiac myofibrils, showing that the fast force decay upon increase in [P] results from so-called sarcomere 'give'. The slow force rise upon decrease in [P] was found to better reflect overall sarcomeres cross-bridge kinetics and its [P] dependence, suggesting that the force generation coupled to P release cannot be separated from the rate-limiting transition. The reasons for the different conclusions achieved in fibre and myofibril studies are re-examined as the recent findings on cardiac myofibrils have fundamental consequences for the coupling between P release, rate-limiting steps and force generation. The implications from P-induced force kinetics of myofibrils are discussed in combination with historical and recent models of the cross-bridge cycle.

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Kinetic Mechanism of Ca2+-controlled Changes of Skeletal Troponin I in Psoas Myofibrils

Cited by 5 publications

References 40 publications

Structural dynamics of troponin during activation of skeletal muscle

Structural dynamics of troponin during activation of skeletal muscle

The Dilated Cardiomyopathy-Causing Mutation ACTC E361G in Cardiac Muscle Myofibrils Specifically Abolishes Modulation of Ca 2+ Regulation by Phosphorylation of Troponin I

Kinetic coupling of phosphate release, force generation and rate-limiting steps in the cross-bridge cycle

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