title: Dual regulatory functions of the thin filament revealed by replacement of the troponin I inhibitory peptide with a linker.
abstract: Striated muscles are relaxed under low Ca(2+) concentration conditions due to actions of the thin filament protein troponin. To investigate this regulatory mechanism, an 11-residue segment of cardiac troponin I previously termed the inhibitory peptide region was studied by mutagenesis. Several mutant troponin complexes were characterized in which specific effects of the inhibitory peptide region were abrogated by replacements of 4-10 residues with Gly-Ala linkers. The mutations greatly impaired two of troponin's actions under low Ca(2+) concentration conditions: inhibition of myosin subfragment 1 (S1)-thin filament MgATPase activity and cooperative suppression of myosin S1-ADP binding to thin filaments with low myosin saturation. Inhibitory peptide replacement diminished but did not abolish the Ca(2+) dependence of the ATPase rate; ATPase rates were at least 2-fold greater when Ca(2+) rather than EGTA was present. This residual regulation was highly cooperative as a function of Ca(2+) concentration, similar to the degree of cooperativity observed with WT troponin present. Other effects of the mutations included 2-fold or less increases in the apparent affinity of the thin filament regulatory Ca(2+) sites, similar decreases in the affinity of troponin for actin-tropomyosin regardless of Ca(2+), and increases in myosin S1-thin filament ATPase rates in the presence of saturating Ca(2+). The overall results indicate that cooperative myosin binding to Ca(2+)-free thin filaments depends upon the inhibitory peptide region but that a cooperatively activating effect of Ca(2+) binding does not. The findings suggest that these two processes are separable and involve different conformational changes in the thin filament.
authors.affiliation: Department of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612, USA.
authors.forename: Julie Mouannes
authors.initials: JM
authors.lastname: Kozaili
authors.forename: Daniel
authors.initials: D
authors.lastname: Leek
authors.forename: Larry S
authors.initials: LS
authors.lastname: Tobacman
mesh_terms: D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D004533:Egtazic Acid / Q000737:chemistry; D006801:Humans; D054792:Muscle, Striated / Q000737:chemistry / Q000378:metabolism; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019210:Troponin I / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D015879:Myosin Subfragments; D010455:Peptides; D014335:Tropomyosin; D019210:Troponin I; D004533:Egtazic Acid; D002118:Calcium