Cardiac Muscle Activation Blunted by a Mutation to the Regulatory Component, Troponin T

Minae Kobayashi,Edward P Debold,Matthew A Turner,Tomoyoshi Kobayashi

doi:10.1074/jbc.m113.494096

Minae Kobayashi, Edward P Debold + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m113.494096

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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.

Highlights

Striated muscle contraction is regulated by Ca2ϩ binding to troponin
Upon Ca2ϩ binding to the regulatory site(s) of troponin C (TnC), a hydrophobic patch is exposed [4] and binds the switch region of troponin I (TnI); a series of conformational transitions occurs among components in the Tn complex, which causes the release of TnI from the actin surface and the movement of Tm to the inner domain of actin [5]
We acknowledge that a number of previous studies have described the effects of DCM-linked mutations in this region of cardiac TnT (cTnT) (18 –20), ours is the first mechanistic study linking the mutation in the H1(T2) region to the attenuation of the Ca2ϩ activation of the thin filament by utilizing biochemical methods including a solution ATPase activity assay, Ca2ϩ binding and S1-ADP binding measurements, and the in vitro motility assay

Summary

Background

Striated muscle contraction is regulated by Ca2ϩ binding to troponin. Results: A mutation at position 205 of cardiac troponin T drastically attenuates the Ca2ϩ activation of the thin filament by altering its properties. Upon Ca2ϩ binding to the regulatory site(s) of TnC, a hydrophobic patch is exposed [4] and binds the switch region of TnI; a series of conformational transitions occurs among components in the Tn complex, which causes the release of TnI from the actin surface and the movement of Tm to the inner domain of actin [5]. This leads to a partial exposure of the high affinity myosin-binding site(s) on the actin. The Ca2ϩ sensitivity of the actomyosin interaction requires the presence of TnT, revealing its critical role in regulating actomyosin inter-

The abbreviations used are

RESULTS

DISCUSSION