Structural changes in troponin in response to Ca ²⁺ and myosin binding to thin filaments during activation of skeletal muscle

Abstract

Contraction of skeletal and cardiac muscle is regulated by Ca 2؉ -dependent structural changes in troponin that control the interaction between myosin and actin. We measured the orientations of troponin domains in skeletal muscle fibers using polarized fluorescence from bifunctional rhodamine probes on the C and E helices of troponin C. The C helix, in the regulatory head domain, tilts by Ϸ30°when muscle is activated in physiological conditions, with a Ca 2؉ -sensitivity similar to that of active force. Complete inhibition of active force did not affect C-helix orientation, and binding of rigor myosin heads did not affect its orientation at saturating [Ca 2؉ ]. The E helix, in the IT arm of troponin, tilted by Ϸ10°on activation, and this was reduced to only 3°when active force was inhibited. Binding of rigor myosin heads produced a larger tilt of the E helix. Thus, in situ, the regulatory head acts as a pure Ca 2؉ -sensor, whereas the IT arm is primarily sensitive to myosin head binding. The polarized fluorescence data from active muscle are consistent with an in vitro structure of the troponin core complex in which the D and E helices of troponin C are collinear. The present data were used to orient this structure in the fiber and suggest that the IT arm is at Ϸ30°to the filament axis in active muscle. In relaxed muscle, the IT arm tilts to Ϸ40°but the D/E helix linker melts, allowing the regulatory head to tilt through a larger angle.

Structural dynamics of troponin during activation of skeletal muscle

“…However, in physiological conditions the steep cooperativity of thin filament activation is not dependent on myosin binding in the steady state (6,36,37). The present results extend this conclusion to the time domain; the dominant components of thin filament activation (phases 1 and 2) are much faster than myosin head binding, and are not affected by its presence (Figs.…”

“…4A, dark green bar and Table S1), or on <P 4 > (Fig. 4A, light green bar), in agreement with previous results (6). In contrast, the difference between <P 2 > values for the E helix in activating and relaxing conditions was reduced to 71 ± 7% (mean ± SD; n = 4) (Fig.…”

“…4A, dark magenta bar and Table S1) of the change during activation in control conditions, similar to that for <P 4 > (77 ± 16%) (Fig. 4A, light magenta bar), indicating that part of the orientation change in the E helix on activation is associated with myosin binding to actin in force generating states (6). No force was generated when Ca 2+ was released from caged Ca in the presence of 0.1 mM BTS (Fig.…”

“…Before the photolysis the fiber was incubated in preactivating solution for ∼5 min and then transferred to the caged calcium solution for another 5 min before UV flash at 12°C. Fiber free [Ca 2+ ] before and after the flash was estimated from the known relationship with <P 2 > and <P 4 > for the TnC probes (6). Before the flash, pCa was set to 6.8 for each fiber by adding to the caged calcium solution the amount of Ca 2+ needed to induce a 5-10% change toward the values of the order parameters during maximal activation.…”

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

“…However, the relative orientation of the ITarm to actin filament axis is more sensitive to strong cross-bridge attachment than to Ca 2+ -binding to the regulatory sites [41]. Moreover, recent data indicate that the IT-arm region is involved in the cross-bridge dependent activation of myofilaments [21].…”