title: Activation dependence and kinetics of force and stiffness inhibition by aluminiofluoride, a slowly dissociating analogue of inorganic phosphate, in chemically skinned fibres from rabbit psoas muscle.
abstract: 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 Ca(2+)-dependence of the kinetics of inhibitor dissociation and the kinetics of actomyosin 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 the kinetic properties of crossbridges attached to actin; sarcomere length was assessed with HeNe laser diffraction. During maximum Ca(2+)-activation at physiological ionic strength (pCa 4.0, 0.2 M gamma/2), stiffness exhibited a steep dependence on the rate of stretch; aluminiofluoride inhibition at pCa 4.0 (0.2 M gamma/2) resulted in an overall decrease 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 of the stiffness-speed relation was reduced during aluminiofluoride inhibition of activated fibres. Relaxation of inhibited fibres (pCa 9.2, 0.2 M gamma/2) resulted in aluminiofluoride being 'trapped' and was accompanied by a further decrease in stiffness at all rates of stretch which was comparable to that found in control relaxed fibres. In relaxed, low ionic strength conditions (pCa 9.2, 0.02 M gamma/2) which promote weak crossbridge binding, stiffness at all rates of stretch was significantly inhibited by aluminiofluoride 'trapped' in the fibre. To determine the Ca(2+)-dependence of inhibitor dissociation, force was regulated independent of Ca2+ using an activating troponin C (aTnC). Results obtained with a TnC-activated fibres confirmed that there is no absolute requirement for Ca2+ for recovery from force inhibition by inorganic phosphate analogues in skinned fibres; the only requirement is thin filament activation which enables active crossbridge cycling. These results indicate that aluminiofluoride preferentially inhibits rapid equilibrium or weak crossbridge attachment to actin, that aluminiofluoride-bound crossbridges attach tightly to the activated thin filament, and that, at maximal (or near-maximal) activation, crossbridge attachment to actin prior to inorganic phosphate analogue dissociation is the primary event regulated by Ca2+.
authors.affiliation: Department of Radiology, University of Washington, Seattle 98195.
authors.forename: P B
authors.initials: PB
authors.lastname: Chase
authors.forename: D A
authors.initials: DA
authors.lastname: Martyn
authors.forename: J D
authors.initials: JD
authors.lastname: Hannon
mesh_terms: D000205:Actomyosin / Q000187:drug effects* / Q000378:metabolism; D017607:Aluminum Compounds / Q000494:pharmacology*; D000818:Animals; D001608:Beryllium / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology / Q000502:physiology; D005459:Fluorides / Q000494:pharmacology*; D007700:Kinetics; D009119:Muscle Contraction / Q000187:drug effects*; D009126:Muscle Relaxation / Q000187:drug effects; D009218:Myosins / Q000378:metabolism; D010710:Phosphates / Q000502:physiology; D016658:Psoas Muscles / Q000187:drug effects / Q000502:physiology; D011817:Rabbits; D014336:Troponin / Q000378:metabolism; D019209:Troponin C; D014638:Vanadates / Q000494:pharmacology
chemical_list: D017607:Aluminum Compounds; D010710:Phosphates; D014336:Troponin; D019209:Troponin C; D014638:Vanadates; C035982:beryllium fluoride; D000205:Actomyosin; D009218:Myosins; D001608:Beryllium; D005459:Fluorides; D002118:Calcium; C032311:aluminum fluoride