The Positively Charged C-Terminal Region of Human Skeletal Troponin T Retards Activation and Decreases Calcium Sensitivity

Abstract

Calcium binding to troponin C (TnC) activates striated muscle contraction by removing TnI (troponin I) from its inhibitory site on actin. Troponin T (TnT) links TnI with tropomyosin, causing tropomyosin to move from an inhibitory position on actin to an activating position. Positive charges within the C-terminal region of human cardiac TnT limit Ca 2+ activation. We now show that the positively charged region of TnT has an even larger impact on skeletal muscle regulation. We prepared one variant of human skeletal TnT that had the C-terminal 16 residues truncated (Δ16) and another with an added C-terminal Cys residue and Ala substituted for the last 6 basic residues (251C-HAHA). Both mutants reduced (based on S1 binding kinetics) or eliminated (based on acrylodan-tropomyosin fluorescence) the first inactive state of actin at <10 nM free Ca 2+ . 251C-HAHA-TnT and Δ16-TnT mutants greatly increased ATPase activation at 0.2 mM Ca 2+ , even without high-affinity cross-bridge binding. They also shifted the force−pCa curve of muscle fibers to lower Ca 2+ by 0.8−1.2 pCa units (the larger shift for 251C-HAHA-TnT). Shifts in force−pCa were maintained in the presence of para-aminoblebbistatin. The effects of modification of the C-terminal region of TnT on the kinetics of S1 binding to actin were somewhat different from those observed earlier with the cardiac analogue. In general, the C-terminal region of human skeletal TnT is critical to regulation, just as it is in the cardiac system, and is a potential target for modulating activity.

Hypertrophic Cardiomyopathy Mutations of Troponin Reveal Details of Striated Muscle Regulation

“…That is, with cardiac troponin, the rate of binding to actin in the active state (M state) is faster than to actin in the inactive-Ca 2+ -bound state (C state) ( Johnson et al, 2019 ). That difference was not observed in the case of skeletal troponin ( Lopez Davila et al, 2020 ).…”

“…Many of these results were recapitulated in skeletal troponin-tropomyosin using a skeletal version 251C-HAHA-TnT or Δ16 skeletal TnT ( Lopez Davila et al, 2020 ). Effects of the loss of charges at the C-terminal region of skeletal TnT included increased ATPase activity at low and high Ca 2+ , loss of the acrylodan tropomyosin fluorescence increase in transitioning to the B state, and an increase in Ca 2+ sensitivity by 0.8–1.2 pCa units.…”

Troponin Variants as Markers of Skeletal Muscle Health and Diseases

“…The Leu 203 truncation mutant retains Tm-binding capacity but lacks TnI and TnC binding sites to also result in complete loss of function ( Amarasinghe et al, 2016 ). Slow TnT mutations that cause deletion of the C-terminal 14 amino acids have been reported to cause ANM-like myopathies in compound heterozygotes with some of the above mutations ( van der Pol et al, 2014 ; Petrucci et al, 2021 ), confirming the functional importance of the C-terminal end segment of TnT ( Lopez Davila et al, 2020 ).…”

Cytotoxicity of snake venom Lys49 PLA2-like myotoxin on rat cardiomyocytes ex vivo does not involve a direct action on the contractile apparatus

“…After chemical permeabilization both ends of the isolated cardiomyocytes were attached to a force transducer and a length driver such that force transients at different free Ca 2+ concentration (pCa) could be measured. Isometric force and the rate constant of isometric force redevelopment after a short period of isotonic shortening ( k TR ) were measured by cycling the cell between isometric steady state contraction and short periods of unloaded isotonic shortening with subsequent re-stretch to the initial (isometric) sarcomere length 35 – 37 . Maximum active isometric force (F max ) at every pCa was obtained by subtracting passive force from the total force, i.e.…”

“…AmBleb is a derivative of blebbistatin 38 , 39 that prevents myosin from entering the force-producing states and thus inhibits force development of striated muscle 36 , 37 . Because force development induces shortening, this treatment permits examination of the cardiomyocytes during and after Mt-II exposure while conserving their structure, since the observed Mt-II-induced hypercontraction is avoided.…”