Alanine or aspartic acid substitutions at serine23/24 of cardiac troponin I decrease thin filament activation, with no effect on crossbridge detachment kinetics.
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 Ca(2+)-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.
Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology (VCAPP), Washington State University, Pullman, WA 99164-6520, USA.
Ranganath
R
Mamidi
Sampath K
SK
Gollapudi
Sri Lakshmi
SL
Mallampalli
Murali
M
Chandra
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007700:Kinetics; D008297:Male; D009119:Muscle Contraction; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism* / Q000502:physiology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009210:Myofibrils / Q000378:metabolism; D010766:Phosphorylation; D051381:Rats; D012694:Serine / Q000235:genetics*; D019210:Troponin I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019210:Troponin I; D001224:Aspartic Acid; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D000409:Alanine; D002118:Calcium