Kinetic Mechanism of Ca 2+-Induced Conformational Changes of Skeletal Muscle Troponin I in Rabbit Psoas Myofibrils

Abstract

The kinetics of Ca2+-controlled conformational changes of the inhibitory subunit (sTnI) of the heterotrimeric skeletal troponin complex were determined by rapid stopped-flow at 10 °C. Conformational changes were probed by fluorescent labelling of sTnI at Cys134 located in between the second actin binding site of sTnI that interacts with actin-tropomyosin at low [Ca2+] and the switch peptide of sTnI that interacts with sTnC at high [Ca2+]. The kinetics was analysed for the sTn-complex in isolation and after its incorporation into rabbit psoas myofibrils. The rapid increase of [Ca2+] to pCa 4.5 induced biphasic fluorescence transients with similar rate constants of k+Ca1.phase ∼800 s−1 and k+Ca2.phase ∼100 s−1 in both preparations. Incorporation changed the polarity of the faster phase but not the polarity of the slower phase. Rapid reduction of [Ca2+] resulted in a monophasic fluorescence change whose rate constant k-Ca was 1.5 s−1 for isolated and 12 s−1 for incorporated sTn-complex. Thus, incorporation of the sTn-complex into the sarcomere increases the off-rate ∼8-fold while leaving the on-rate unaffected. The values of k+Ca2.phase and k-Ca determined in our myofibrils experiments are very similar to the values of kON and kOFF reported by Brenner & Chalovich (Biophys J. 1999 77:2692-708). They observed monophasic kinetics for sTnI labelled at Cys134 incorporated into skinned rabbit psoas fibers. In contrast to our experiments, they triggered sTnI-kinetics mechanically, i.e. by rapidly changing the number of force-generating cross-bridges. The faster conformational change in sTnI that is only observed in our Ca2+-triggered experiments is therefore likely associated with the Ca2+-binding process while the slower phase reports the conformational change of sTnI involved in force regulation. Supported by the Center of Molecular Medicine Cologne (CMMC-A6) and the DFG (SFB612-A2).