title: Cardiac muscle activation blunted by a mutation to the regulatory component, troponin T.
abstract: The striated muscle thin filament comprises actin, tropomyosin, and troponin. The Tn complex consists of three subunits, troponin C (TnC), troponin I (TnI), and troponin T (TnT). TnT may serve as a bridge between the Ca(2+) sensor (TnC) and the actin filament. In the short helix preceding the IT-arm region, H1(T2), there are known dilated cardiomyopathy-linked mutations (among them R205L). Thus we hypothesized that there is an element in this short helix that plays an important role in regulating the muscle contraction, especially in Ca(2+) activation. We mutated Arg-205 and several other amino acid residues within and near the H1(T2) helix. Utilizing an alanine replacement method to compare the effects of the mutations, the biochemical and mechanical impact on the actomyosin interaction was assessed by solution ATPase activity assay, an in vitro motility assay, and Ca(2+) binding measurements. Ca(2+) activation was markedly impaired by a point mutation of the highly conserved basic residue R205A, residing in the short helix H1(T2) of cTnT, whereas the mutations to nearby residues exhibited little effect on function. Interestingly, rigor activation was unchanged between the wild type and R205A TnT. In addition to the reduction in Ca(2+) sensitivity observed in Ca(2+) binding to the thin filament, myosin S1-ADP binding to the thin filament was significantly affected by the same mutation, which was also supported by a series of S1 concentration-dependent ATPase assays. These suggest that the R205A mutation alters function through reduction in the nature of cooperative binding of S1.
authors.affiliation: From the Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, Illinois 60612 and. Electronic address: minaek@uic.edu.
authors.forename: Minae
authors.initials: M
authors.lastname: Kobayashi
authors.affiliation: the Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Edward P
authors.initials: EP
authors.lastname: Debold
authors.affiliation: the Department of Kinesiology, University of Massachusetts, Amherst, Massachusetts 01003.
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Turner
authors.affiliation: From the Department of Physiology and Biophysics, Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, Illinois 60612 and.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Kobayashi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D051379:Mice; D020125:Mutation, Missense; D009206:Myocardium / Q000737:chemistry / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D020107:Troponin T / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D020107:Troponin T; D009218:Myosins; D002118:Calcium
keywords: Actin; Calcium; Muscle; Myosin; Troponin
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