title: Modulation of troponin C affinity for the thin filament by different cross-bridge states in skinned skeletal muscle fibers.
abstract: In vertebrate skeletal muscle, the C-domain of troponin C (TnC) serves as an anchor; the N-domain regulates the position of troponin-tropomyosin on the thin filament after changes in intracellular Ca2+. Another type of thin-filament regulation is provided by cross-bridges. In this study, we use skinned fibers reconstituted with chicken recombinant TnC (rTnC) to examine TnC-thin filament affinity when cross-bridges containing different ligands are formed. Dissociation and equilibrium binding of apo-TnC (i.e., lacking divalent cations) under different conditions were monitored by a standard test for maximum tension (P (o)). After 10 min in low-Mg2+ relaxing solution, rTnC dissociation (i.e., tension loss) was 80% vs only 45% in rigor. In rigor, adding myosin subfragment 1 (S1) reduced dissociation approximately twofold, whereas stretching to reduce filament overlap increased dissociation to nearly the value for relaxed fibers. Dissociation of rTnC after addition of Pi or MgADP to form A.M.Pi or A.M.ADP cross-bridges was significantly greater than with rigor (A.M) bridges. The increase in P (o) during equilibration with different concentrations of rTnC showed that the affinity for rTnC binding to the thin filament increased progressively with stronger cross-bridges: rTnC concentrations for half-maximal reconstitution (K (0.5)) were 8.1, 3.7, 2.9, and 1.1 microM for A + M.ADP.Pi, A.M.Pi, A.M, and A.M + S1. Cross-bridges containing MgADP(-) (A.M.ADP) were also less effective than rigor bridges in promoting rTnC binding. We conclude that cross-bridge state and number both modulate TnC affinity for the thin filament and that the TnC C-domain is a central element in this pathway.
authors.affiliation: Instituto de Bioquímica Médica, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, RJ, 21941-590, Brazil.
authors.forename: José Renato
authors.initials: JR
authors.lastname: Pinto
authors.forename: Tiago
authors.initials: T
authors.lastname: Veltri
authors.forename: Martha M
authors.initials: MM
authors.lastname: Sorenson
mesh_terms: D000205:Actomyosin / Q000737:chemistry; D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D002118:Calcium / Q000737:chemistry; D002413:Cations, Divalent / Q000737:chemistry; D002645:Chickens; D003599:Cytoskeleton / Q000378:metabolism*; D007202:Indicators and Reagents; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D012996:Solutions; D019209:Troponin C / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D007202:Indicators and Reagents; D008024:Ligands; D010710:Phosphates; D012996:Solutions; D019209:Troponin C; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000205:Actomyosin; D009218:Myosins; D008274:Magnesium; D002118:Calcium