Kinetics of a Ca²⁺-sensitive cross-bridge state transition in skeletal muscle fibers: Effects due to variations in thin filament activation by extraction of troponin C

The rate constant of tension redevelopment (k_tr; 1986. Proc. Natl. Acad. Sci. USA. 83:3542-3546) was determined at various levels of thin filament activation in skinned single fibers from mammalian fast twitch muscles. Activation was altered by (a) varying the concentration of free Ca²⁺ in the activating solution, or (b) extracting various amounts of troponin C (TnC) from whole troponin complexes while keeping the concentration of Ca²⁺ constant. TnC was extracted by bathing the fiber in a solution containing 5 mM EDTA, 10 mM HEPES, and 0.5 mM trifluoperazine dihydrochloride. Partial extraction of TnC resulted in a decrease in the Ca²⁺ sensitivity of isometric tension, presumably due to disruption of near-neighbor molecular cooperativity between functional groups (i.e., seven actin monomers plus associated troponin and tropomyosin) within the thin filament. Altering the level of thin filament activation by partial extraction of TnC while keeping Ca²⁺ concentration constant tested whether the Ca²⁺ sensitivity of k_tr results from a direct effect of Ca²⁺ on cross-bridge state transitions or, alternatively, an indirect effect of Ca²⁺ on these transitions due to varying extents of thin filament activation. Results showed that the k_tr-pCa relation was unaffected by partial extraction of TnC, while steady-state isometric tension exhibited the expected reduction in Ca²⁺ sensitivity. This finding provides evidence for a direct effect of Ca²⁺ on an apparent rate constant that limits the formation of force-bearing cross-bridge states in muscle fibers. Further, the kinetics of this transition are unaffected by disruption of near-neighbor thin filament cooperativity subsequent to extraction of TnC. Finally, the results support the idea that the steepness of the steady-state isometric tension-calcium relationship is at least in part due to mechanisms involving molecular cooperativity among thin filament regulatory-proteins.