Ca2+ regulation of rabbit skeletal muscle thin filament sliding:: Role of cross-bridge number

We investigated how strong cross-bridge number affects sliding speed of regulated Ca2+-activated, thin filaments. First, using in vitro motility assays, sliding speed decreased nonlinearly with reduced density of heavy meromyosin (HMM) for regulated (and unregulated) F-actin at maximal Ca2+. Second, we varied the number of Ca2+-activatable troponin complexes at maximal Ca2+ using mixtures of recombinant rabbit skeletal troponin (WT sTn) and sTn containing sTnC(D27A, D63A), a mutant deficient in Ca2+ binding at both N-terminal, low affinity Ca2+-binding sites (xxsTnC-sTn). Sliding speed decreased nonlinearly as the proportion of WT sTn decreased. Speed of regulated thin. laments varied with pCa when. laments contained WT sTn but. laments containing only xxsTnC-sTn did not move. pCa(50) decreased by 0.12-0.18 when either heavy meromyosin density was reduced to similar to60% or the fraction of Ca2+-activatable regulatory units was reduced to similar to33%. Third, we exchanged mixtures of sTnC and xxsTnC into single, permeabilized fibers from rabbit psoas. As the proportion of xxsTnC increased, unloaded shortening velocity decreased nonlinearly at maximal Ca2+. These data are consistent with unloaded. lament sliding speed being limited by the number of cycling cross-bridges so that maximal speed is attained with a critical, low level of actomyosin interactions.