Measurement of calcium dissociation rates from troponin C in rigor skeletal myofibrils

Abstract

Ca2+ dissociation from the regulatory domain of troponin C may influence the rate of striated muscle relaxation. However, Ca 2+ dissociation from troponin C has not been measured within the geometric and stoichiometric constraints of the muscle fiber. Here we report the rates of Ca 2+ dissociation from the N-terminal regulatory and C-terminal structural domains of fluorescent troponin C constructs reconstituted into rabbit rigor psoas myofibrils using stopped-flow technology. Chicken skeletal troponin C fluorescently labeled at Cys 101, troponin C IAEDANS, reported Ca 2+ dissociation exclusively from the structural domain of troponin C at ~0.37, 0.06, and 0.07/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Ca 2+ dissociation from the regulatory domain was observed utilizing fluorescently labeled troponin C containing the T54C and C101S mutations. Troponin C T54C,C101SMIANS reported Ca 2+ dissociation exclusively from the regulatory domain of troponin C at >1000, 8.8, and 15/s in isolation, in the presence of troponin I and in myofibrils at 15°C, respectively. Interestingly, troponin C T54C,C101SMIANS reported a biphasic fluorescence change upon Ca 2+ dissociation from the N and C-terminal domains of troponin C with rates that were similar to those reported by troponin C T54C,C101SIAANS and troponin C IAEDANS at all levels of the troponin C systems. Furthermore, the rate of Ca 2+ dissociation from troponin C in the myofibrils was similar to the rate of Ca 2+ dissociation measured from the troponin C-troponin I complexes. Since the rate of Ca 2+ dissociation from the regulatory domain of TnC in myofibrils is similar to the rate of skeletal muscle relaxation, Ca 2+ dissociation from troponin C may influence relaxation. © 2011 Little, Tikunova, Norman, Swartz and Davis.