Kinetic mechanism of Ca²⁺-controlled changes of skeletal troponin I in psoas myofibrils

Abstract

Conformational changes in the skeletal troponin complex (sTn) induced by rapidly increasing or decreasing the [Ca(2+)] were probed by 5-iodoacetamidofluorescein covalently bound to Cys-133 of skeletal troponin I (sTnI). Kinetics of conformational changes was determined for the isolated complex and after incorporating the complex into rabbit psoas myofibrils. Isolated and incorporated sTn exhibited biphasic Ca(2+)-activation kinetics. Whereas the fast phase (k(obs)∼1000 s(-1)) is only observed in this study, where kinetics were induced by Ca(2+), the slower phase resembles the monophasic kinetics of sTnI switching observed in another study (Brenner and Chalovich. 1999. Biophys. J. 77:2692-2708) that investigated the sTnI switching induced by releasing the feedback of force-generating cross-bridges on thin filament activation. Therefore, the slower conformational change likely reflects the sTnI switch that regulates force development. Modeling reveals that the fast conformational change can occur after the first Ca(2+) ion binds to skeletal troponin C (sTnC), whereas the slower change requires Ca(2+) binding to both regulatory sites of sTnC. Incorporating sTn into myofibrils increased the off-rate and lowered the Ca(2+) sensitivity of sTnI switching. Comparison of switch-off kinetics with myofibril force relaxation kinetics measured in a mechanical setup indicates that sTnI switching might limit the rate of fast skeletal muscle relaxation.

Figure 1

Effect of labeling on the kinetics of sTn and incorporation into the sarcomere. (A) Effect of changing the label on kinetics. Myofibrils having incorporated either sTn^IAF or sTn^NBD are mixed at time = 0 with high [Ca²⁺] (pCa 4.6). (B) Effect of IAF labeling on the kinetics of Ca²⁺ dissociation from isolated sTn probed by Quin-2 fluorescence. (C–E) Localization of sTn^IAF in the sarcomere resolved by confocal microscopy. Green: sTn^IAF emission, red: Z discs marked with Alexa 532/anti-α-actinin.

Figure 2

Biphasic kinetics of sTn^IAF following a rise in [Ca²⁺] and the origin of fluorescence changes. (A) Percental fluorescence changes upon mixing isolated sTn^IAF with high [Ca²⁺] in the standard and microcuvette, both mixings resulting in pCa 4.6. (B) As in A for sarcomeric sTn^IAF (only standard cuvette). (C) sTnT, sTnI, and sTnC subunits from 100 μg sTn^IAF and 100 μg sTn^NBD are separated on a 10–20% SDS-polyacrylamide gel (Tris-Tricine) and IAF-fluorescence excited by ultraviolet light. (D) Fluorescence transients of myofibrils with incorporated IAF-labeled sTn complexes that have been selectively labeled either on TnC (sTn^TnC-IAF) or on TnI (sTn^TnI-IAF).

Figure 3

Ca²⁺ dependence of sTn^IAF switch-on and its relation to force-development kinetics. (A) Fluorescence transients following mixing isolated sTn^IAF with increasing [Ca²⁺]. (B) As in A but for sarcomeric sTn^IAF. (C) Kinetics of Ca²⁺-induced force development of myofibrils with incorporated sTn^IAF. (D) Ca²⁺ dependence of total fluorescence changes in isolated and sarcomeric sTn^IAF in relation to force. (E) Ca²⁺ dependence of the fluorescence amplitudes of the fast (negative) and slow (positive) phase of sarcomeric sTn^IAF. (F) Ca²⁺ dependence of the rate constants of the fast phase of sarcomeric sTn^IAF. (G) Ca²⁺ dependence of the rate constants of the slow phase of isolated and sarcomeric sTn^IAF. (H) Ca²⁺ dependence of the rate constant of Ca²⁺-induced force development. Note, Hill curves in D–H represent a single fit to the means pooled from all experiments and might deviate slightly from the Hill parameters listed in Table 2 obtained from fitting Hill curves to individual experiments.

Figure 4

Kinetics of Ca²⁺-induced switch-off of sTn^IAF and its relation to force relaxation. (A) Normalized fluorescence changes upon mixing Ca²⁺-activated, isolated sTn^IAF, or Ca²⁺-activated sarcomeric sTn^IAF ± ATP with the rapid Ca²⁺-chelator BAPTA. Note the biphasic transient with sarcomeric sTn^IAF + ATP. (B) Fluorescence changes in sarcomeric sTn^IAF (as in A) compared to the biphasic kinetics of force relaxation. Although the initial slow linear phase of the force decay lasts longer than the fast fluorescence increase of sarcomeric sTn^IAF + ATP, the subsequent slow phases exhibit similar kinetics.