Structural and Kinetic Effects of PKC Phosphorylation and Selected HCM Mutations of cTnI on cTnI-CTnC Interactions within the Reconstituted Thin Filament

Abstract

The switching activity of cardiac troponin I (cTnI) during activation can be modulated by protein kinase C (PKC) dependent phosphorylation and hypertrophic cardiomyopathy (HCM) mutations found in the C-domain of cTnI. In this study, FRET was used to follow the cTnI-cTnC interactions associated with cTnI switching by monitoring changes in the distances from residue 89 of cTnC to residues 151 (inhibitory region-Ir) and 167 (regulatory region-Rr) of cTnI, respectively. PKC pseudo-phosphorylation mutants of cTnI (S43E, S45E and T144E) and three HCM-associated mutants of cTnI (R145G, R145Q and R162W), all containing a marker for FRET, were generated. The structural and kinetic effects of these mutants on cTnI-cTnC interactions were then investigated using steady-state and time-resolved FRET measurements and stopped-flow. Results showed that PKC pseudo-phosphorylation of cTnI decreased the Ca2+-sensitivity of both the cTnI(Ir)- and cTnI(Rr)-cTnC interactions, while the HCM mutants cTnI(R145G), cTnI(R145Q) and cTnI(R163W) increased Ca2+-sensitivity. Ca2+-dissociation induced structural kinetics of the cTnI(Ir)-cTnC interaction was accelerated with cTnI(S43E/S45E) and cTnI(S43E/S45E/T144E), and slowed with cTnI(T144E). The structural kinetics of the cTnI(Rr)-cTnC interaction was decreased in response to all PKC pseudo-phosphorylation combinations. Furthermore, the presence of any of the three HCM mutations slowed the kinetics of Ca2+-dissociation induced structural changes in both cTnI(Ir)- and cTnI(Rr)-cTnC interactions, but to a different extent. These results suggest that different HCM mutations and PKC phosphorylation of cTnI may have unique structural and kinetic effects on cTnI(Ir)- and cTnI(Rr)-cTnC interactions that collectively effect pathophysiology and functional modulation, respectively. Structural effects of the PKC pseudo-phosphorylation mutants and HCM mutants, as well as overall effects when PKC pseudo-phosphorylation and HCM mutations are combined into one mutant, on both cTnI(Ir)- and cTnI(Rr)-cTnC interactions will be also discussed.