P334Molecular effects of the amino acid exchanges R20C and K206Q of cardiac troponin I, linked to familial hypertrophic cardiomyopathy (FHC)

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Abstract

Purpose

Troponin (Tn), the main regulatory protein of thin filaments, mediates calcium-sensitivity of the actin-myosin interaction. Mutations linked to familial hypertrophic cardiomyopathy (FHC) have been found in all domains of cardiac TnI, the inhibitory subunit of the heterotrimeric troponin complex.

Purpose

Phosphorylation of S22, 23 in the cardiac specific N-terminal arm of cTnI by PKA is known to reduce the Ca2+- sensitivity of the actin-myosin (AM) interaction and to enhance the relaxation rate of myofibrils. The FHC inducing amino acid exchange R20C is located within the PKA consensus sequence in the cTnI N-terminus.

Purpose

The C-terminal region of cTnI seems to stabilize the Ca2+-activated state of the thin filament. The FHC inducing amino acid exchange in this part of cTnI, K206Q, increases the Ca2+-sensitivity of AM S1-ATPase activity as well as its maximum activity and filament sliding velocity.

Purpose

We investigated the impact of R20C and K206Q on PKA phosphorylation effects, on protein interactions in the thin filament and on Ca2+-sensitivity of AM S1-ATPase activity.

Methods

To study protein interactions we used peptide arrays, co-sedimentation and SPR spectroscopy. Phosphorylation studies were done by treating cTnI with PKA catalytic subunit and subsequently separating by IEF, as well as by replacement of serines 22,23 by aspartic acid. AM S1-ATPase activity was measured in an enzyme-coupled assay by detection of UV-absorption.

Results

In R20C, PKA dependent phosphorylation was highly decreased. The interaction of the N-terminus with cTnC was unaltered in peptide arrays, but binding to the thin filament was decreased by 30%. Thin filaments containing R20C differed from WT both in the Ca2+-sensitivity of the AM S1-ATPase and its maximal activity. Replacement of S22,23 by Asp lead to major changes in all kinetic parameters and increased thin filament binding.

Results

The interaction of the C-terminus with actin is completely abolished in K206Q. Accordingly, decreased thin filament binding of K206Q by 31% (P < 0,05) was found in co-sedimentation. Replacement of S22,23 by Asp resulted in a further decrease of binding. The dissociation constant of 4.1 μM for the interaction between K206Q-containing troponin and actin, measured by SPR, was significantly decreased compared to WT (4.7 μM).

Conclusions

The main pathogenic effect of R20C might be due to the impaired PKA dependent phosphorylation. Replacement of S22,23 by Asp probably causes structural distortions in the cTnI N-terminus in this mutant.

Conclusions

K206Q destabilizes Tn/actin interaction, which might explain the effects on calcium sensitivity of thin filament activation.

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