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

► 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.