Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

Ranganath Mamidi; Sampath K Gollapudi; Sri Lakshmi Mallampalli; Murali Chandra

doi:10.1016/j.abb.2012.05.024

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Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

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Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics

Ranganath Mamidi, Sampath K Gollapudi, Sri Lakshmi Mallampalli and Murali Chandra

Archives of biochemistry and biophysics, Vol.525(1), pp.1-8

09/01/2012

DOI: https://doi.org/10.1016/j.abb.2012.05.024

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https://hdl.handle.net/2376/103459

PMCID: PMC3405180

PMID: 22684024

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Abstract

Phosphorylation

Reconstitution

Cardiac troponin I

Homologous proteins

Substitutions

► Homologous proteins were used for reconstitution in our functional assay. ► Ala/Asp substitutions were introduced at Ser23/24 of cardiac troponin I (cTnI). ► Protein kinase A phosphorylation was mimicked using Asp substitutions in cTnI. ► Ala/Asp substitutions at Ser23/24 of cTnI decreased the thin filament activation. ► Ca2+ sensitivity decreased with Asp substitutions, but not with Ala substitutions. Ala/Asp substitutions at Ser23/24 have been employed to investigate the functional impact of cardiac troponin I (cTnI) phosphorylation by protein kinase A (PKA). Some limitations of previous studies include the use of heterologous proteins and confounding effects arising from phosphorylation of cardiac myosin binding protein-C. Our goal was to probe the effects of cTnI phosphorylation using a homologous assay, so that altered function could be solely attributed to changes in cTnI. We reconstituted detergent-skinned rat cardiac papillary fibers with homologous rat cardiac troponin subunits to study the impact of Ala and Asp substitutions at Ser23/24 of rat cTnI (RcTnI S23A/24A and RcTnI S23D/24D). Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼36% decrease in Ca2+-activated maximal tension. Both RcTnI S23A/24A and RcTnI S23D/24D showed a ∼18% decrease in ATPase activity. Muscle fiber stiffness measurements suggested that the decrease in thin filament activation observed in RcTnI S23A/24A and RcTnI S23D/24D was due to a decrease in the number of strongly-bound crossbridges. Another major finding was that Ala and Asp substitutions in cTnI did not affect crossbridge detachment kinetics.

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Title: Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics
Creators: Ranganath Mamidi
Sampath K Gollapudi
Sri Lakshmi Mallampalli
Murali Chandra
Publication Details: Archives of biochemistry and biophysics, Vol.525(1), pp.1-8
Academic Unit: Integrative Physiology and Neuroscience, Department of
Publisher: Elsevier Inc
Identifiers: 99900546673901842
Language: English
Resource Type: Journal article