The Cardiomyopathy Mutation, R146G Troponin I, Stabilizes the Intermediate “C” State of Regulated Actin under High- and Low-Free Ca2+ Conditions

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• 作者：Dylan Johnson ; Mohit C. Mathur ; Tomoyoshi Kobayashi ; Joseph M. Chalovich
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：August 16, 2016
• 年：2016
• 卷：55
• 期：32
• 页码：4533-4540
• 全文大小：365K
• 年卷期：0
• ISSN：1520-4995

文摘

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 Ca²⁺ concentrations. We show, from measurements of the k_cat for actin-activated ATPase activity at saturating Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ 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.