ACTIVATION DEPENDENCE AND KINETICS OF FORCE AND STIFFNESS INHIBITION BY ALUMINIOFLUORIDE, A SLOWLY DISSOCIATING ANALOG OF INORGANIC-PHOSPHATE, IN CHEMICALLY SKINNED FIBERS FROM RABBIT PSOAS MUSCLE

To examine the mechanism by which aluminiofluoride, a tightly binding analogue of inorganic phosphate, inhibits force in single, chemically skinned fibres from rabbit psoas muscle, we measured the Ca2+-dependen ce of the kinetics of inhibitor dissociation and the kinetics of actom yosin interactions when aluminiofluoride was bound to the crossbridges . The relation between stiffness and the speed of stretch during small amplitude ramp stretches (<5 nm per h.s.) was used to characterize th e kinetic properties of crossbridges attached to actin; sarcomere leng th was assessed with HeNe laser diffraction. During maximum Ca2+-activ ation at physiological ionic strength (pCa 4.0, 0.2 M Gamma/2), stiffn ess exhibited a steep dependence on the rate of stretch; aluminiofluor ide inhibition at pCa 4.0 (0.2 M Gamma/2) resulted in an overall decre ase in stiffness, with stiffness at high rates of stretch (10(3)-10(4) nm per h.s. per s) being disproportionately reduced. Thus the slope o f the stiffness-speed relation was reduced during aluminiofluoride inh ibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 M Gamma/2) resulted in aluminiofluoride being 'trapped' and was ac companied by a further decrease in stiffness at all rates of stretch w hich was comparable to that found in control relaxed fibres. In relaxe d, low ionic strength conditions (pCa 9.2, 0.02 M Gamma/2) which promo te weak crossbridge binding, stiffness at all rates of stretch was sig nificantly inhibited by aluminiofluoride 'trapped' in the fibre. To de termine the Ca2+-dependence of inhibitor dissociation, force was regul ated independent of Ca2+ using an activating troponin C (aTnC). Result s obtained with aTnC-activated fibres confirmed that there is no absol ute requirement for Ca2+ for recovery from force inhibition by inorgan ic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These r esults indicate that aluminiofluoride preferentially inhibits rapid eq uilibrium or weak crossbridge attachment to actin, that aluminiofluori de-bound crossbridges attach tightly to the activated thin filament, a nd that, at maximal (or near-maximal) activation, crossbridge attachme nt to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca2+.