Crossbridge-mediated Activation of Rabbit Skeletal Muscle Myofibrillar ATPase: a Role for the Calcium Binding Domains of Troponin C

Abstract

Activation of the thin filament in striated muscle is a cooperative process requiring both the binding of Ca2+ to troponin C (TnC) and the binding of myosin crossbridges to actin. The aim of this study was to assess the role of TnC domains in the crossbridge-mediated activation of rabbit skeletal muscle myofibrils in the absence of Ca2+. Activation of myofibrillar ATPase was produced by addition of varying concentrations of myosin S1 modified by N-ethylmaleimide (NEM-S1), which facilitates crossbridge cycling by forcing tropomyosin into the open position on the filament. Comparisons were made of native myofibrils, myofibrils from which TnC was extracted, and myofibrils reconstituted with either a TnC mutant (TnC48-82) in which Ca2+ activation was blocked by a disulfide bond in the N-terminal domain (Grabarek, et al, Nature, 345:132,1990) or a proteolytic fragment of TnC (TR2C) lacking the N-terminal Ca2+- binding domain (Grabarek, et al, J. Biol. Chem. 265:13121, 1981). The ATPase activity of native myofibrils was increased ∼170% by the addition of NEM-S1 (2-4μM). Following extraction of TnC the addition of the same concentrations of NEM-S1 produced ∼60% activation. In both cases higher concentrations of NEM-S1 produced no further increase in activation. With the addition of either TnC48-82 or TR2C the degree of activation was higher (70-100%), but required higher NEM-S1 concentrations (4-8μM). These results suggest that both domains of TnC play a role in facilitating optimal crossbridge-mediated activation of the thin filament, presumably by providing alternative binding sites for troponin I.