title: The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate "C" State of Regulated Actin under High- and Low-Free Ca(2+) Conditions.
abstract: The R146G mutation of troponin I (TnI) is associated with hypertrophic cardiomyopathy in humans. Earlier data pointed to stabilization of the intermediate, C state, of actin-tropomyosin-troponin by this mutant. Because cardiac disorders appear to be linked to changes in regulated actin distributions, we determined the extent to which the R146G TnI mutant alters the distribution of states at low and high Ca(2+) concentrations. We show, from measurements of the kcat for actin-activated ATPase activity at saturating Ca(2+) concentrations, that R146G TnI reduced the population of the active, M, state to 25% of the wild-type level. Together with acrylodan-tropomyosin fluorescence measurements of the B state, it appeared that the C state was populated at ∼91% of the total for the R146G TnI-containing actin filaments. The C state was also more heavily populated at low Ca(2+) concentrations. Acrylodan-tropomyosin fluorescence changes showed a large diminution in the inactive state value relative to the wild-type value without a comparable increase in the active state. Furthermore, the rate of binding of rigor S1 to pyrene-labeled actin filaments containing R146G TnI was faster than the rate of binding to wild-type filaments at low free Ca(2+) concentrations. These results indicate that the inhibitory region of TnI affects the B-C and M-C equilibria of actin-tropomyosin-troponin. The observation that a mutation in the inhibitory region affects the M-C equilibrium may point to a novel regulatory interaction.
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University , Greenville, North Carolina 27834, United States.|Department of Chemistry, East Carolina University , Greenville, North Carolina 27858-4353, United States.
authors.forename: Dylan
authors.initials: D
authors.lastname: Johnson
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Mohit C
authors.initials: MC
authors.lastname: Mathur
authors.affiliation: Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois , Chicago, Illinois 60612-7342, United States.
authors.forename: Tomoyoshi
authors.initials: T
authors.lastname: Kobayashi
authors.affiliation: Department of Biochemistry and Molecular Biology, Brody School of Medicine, East Carolina University , Greenville, North Carolina 27834, United States.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Chalovich
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D009202:Cardiomyopathies / Q000235:genetics* / Q000378:metabolism; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D051379:Mice; D009154:Mutation; D055550:Protein Stability; D011817:Rabbits; D019210:Troponin I / Q000235:genetics*
chemical_list: D000199:Actins; D019210:Troponin I; D002118:Calcium