Role of Ca2+ and cross-bridges in skeletal muscle thin filament activationprobed with Ca2+ sensitizers

Thin filament regulation of contraction is thought to involve the binding o f two activating ligands: Ca2+ and strongly bound cross-bridges. The specif ic cross-bridge states required to promote thin filament activation have no t been identified. This study examines the relationship between cross-bridg e cycling and thin filament activation by comparing the results of kinetic experiments using the Ca2+ sensitizers: caffeine and bepridil. In single sk inned rat soleus fibers, 30 mM caffeine produced a leftward shift in the te nsion-pCa relation from 6.03 +/- 0.03 to 6.51 +/- 0.03 pCa units and lowere d the maximum tension to 0.60 +/- 0.01 of the control tension. In addition, the rate of tension redevelopment (k(tr)) was decreased from 3.51 +/- 0.-1 2 s(-1) to 2.70 +/- 0.19 s(-1), and V-max decreased from 1.24 +/- 0.07 to 0.64 +/- 0.02 M.L./s. Bepridil produced a similar shift in the tension-pCa curves but had no effect on the kinetics. Thus bepridil increases the Ca2sensitivity through direct effects on TnC, whereas caffeine has significant effects on the cross-bridge interaction. interestingly, caffeine also prod uced a significant increase in stiffness under relaxing conditions (pCa 9.0 ), indicating that caffeine:induces some strongly bound cross-bridges, even in the absence of Ca2+. The results are interpreted in terms of a model in tegrating cross-bridge cycling with a three-state thin-filament activation model, Significantly, strongly bound, non-tension-producing cross-bridges w ere essential to modeling of complete activation of the thin filament.