Effects of PKA Phosphorylation of Cardiac Troponin I and Strong Crossbridge on Conformational Transitions of the N-Domain of Cardiac Troponin C in Regulated Thin Filaments

Abstract

Regulation of cardiac muscle function is initiated by binding of Ca 2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine tuned by phosphorylation of cTnI. The objective of the present study is to use a new Förster Resonance Energy Transfer-based structural marker to distinguish structural and kinetic effects of Ca 2+ binding, crossbridge interaction and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a doublecysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin and actin. Changes in the distance between Cys13 and Cys51 induced by Ca 2+ binding/dissociation were determined by FRET-sensed Ca 2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca 2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca 2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca 2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes. Keywordsphosphorylation; cardiac troponin C; thin filament; FRET; Ca 2+ activation; kinetics Force development during cardiac muscle contraction is dependent upon the strong interactions between myosin and the actin filament. These interactions are governed by the regulatory

FRET study of the structural and kinetic effects of PKC phosphomimetic cardiac troponin T mutants on thin filament regulation

“…This implies that the mechanism of action for the T204E mutation is completely different than that of T195E and T285E in not significantly impacting the distance between cTnI-Sr and N-cTnC; a plausible T204E structural change that might go undetected by our FRET scheme is a change in C-cTnI–N-cTnC orientation that affects the kinetics associated with I-C switching without disrupting the average position of a functional regions per se , thereby reducing Ca 2+ -sensitivity. Observation of such a kinetic effect was previously made in the case of the effects of PKA-mediated phosphorylation of cTnI on N-cTnC opening [37]. …”

“…To implement FRET in this study, a series of recombinant single cysteine mutants were generated from wild type rat protein clones using approaches similar to those previously reported [10, 13, 37, 38]. Briefly, using the GeneTailor™ Site-Directed Mutagenesis System (Invitrogen, Carlsbad, CA, USA), rat cDNA clones of wild-type cTnC, cTnI and cTnT sub-cloned into the plasmid pSBETa was used as a template DNA to generate single-cysteine cTnC and cTnI mutants and phosphomimetic mutants of cTnT.…”

Enhanced Ca2+ binding of cardiac troponin reduces sarcomere length dependence of contractile activation independently of strong crossbridges

“…This should increase cTnC-cTnI interaction, allowing enhanced movement of Tm and access to myosin binding sites on actin for a given submaximal [Ca 2ϩ ]. It is likely that this is the cause of increased Ca 2ϩ sensitivity of thin filament activation and force development in cardiac muscle containing L48Q cTnC, and it may also reduce the requirement for crossbridges to stabilize the cTnC-cTnI state (7). This effect should be more pronounced at lower [Ca 2ϩ ], where the slower dissociation rate of L48Q cTnC (27,44) should result in Ca 2ϩ being bound to more cTn in thin filaments at any given time.…”

Structural Dynamics of C-domain of Cardiac Troponin I Protein in Reconstituted Thin Filament

“…Reconstitution of cTn complexes from wild-type cTnC, TAMRA-or IAANS-labeled cTnI, and wild-type cTnT was carried out in 6 M urea at a molar ratio of 1.2:1.0:1.2 cTnC:cTnI: cTnT following a procedure reported previously (27). Reconstituted cTn containing one of the modified cTnI mutants was then used together with Tm and actin to form the reconstituted thin filament for use in anisotropy experiments (27).…”

“…Reconstituted cTn containing one of the modified cTnI mutants was then used together with Tm and actin to form the reconstituted thin filament for use in anisotropy experiments (27). cTnI mutants were also incorporated into detergent-skinned muscle fibers (28), and the following experiments were performed to test for any effects on protein stability and functional integrity caused by cTnI mutations and probe modifications: 1) SDS-PAGE and native gel analysis, 2) ATPase activity measurements, and 3) measurement of the force-Ca 2ϩ relationship under steady-state conditions.…”