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

Abstract

FRET was used to investigate the structural and kinetic effects that PKC phosphorylations exert on Ca(2+) and myosin subfragment-1 dependent conformational transitions of the cardiac thin filament. PKC phosphorylations of cTnT were mimicked by glutamate substitution. Ca(2+) and S1-induced distance changes between the central linker of cTnC and the switch region of cTnI (cTnI-Sr) were monitored in reconstituted thin filaments using steady state and time resolved FRET, while kinetics of structural transitions were determined using stopped flow. Thin filament Ca(2+) sensitivity was found to be significantly blunted by the presence of the cTnT(T204E) mutant, whereas pseudo-phosphorylation at additional sites increased the Ca(2+)-sensitivity. The rate of Ca(2+)-dissociation induced structural changes was decreased in the C-terminal end of cTnI-Sr in the presence of pseudo-phosphorylations while remaining unchanged at the N-terminal end of this region. Additionally, the distance between cTnI-Sr and cTnC was decreased significantly for the triple and quadruple phosphomimetic mutants cTnT(T195E/S199E/T204E) and cTnT(T195E/S199E/T204E/T285E), which correlated with the Ca(2+)-sensitivity increase seen in these same mutants. We conclude that significant changes in thin filament Ca(2+)-sensitivity, structure and kinetics are brought about through PKC phosphorylation of cTnT. These changes can either decrease or increase Ca(2+)-sensitivity and likely play an important role in cardiac regulation.

Keywords: Cardiac troponin T; FRET; PKC phosphorylation; Thin filament regulation.

Figure 1

Comparison of AEDANS fluorescence intensity as a function of emission wavelength for either donor-acceptor samples (DA) or donor only samples (D) under various physiological conditions. These representative traces of cTnI(S167C_AEDANS)-cTnC(S89C_DDPM) show substantial FRET quenching compared to donor only control in low Ca²⁺ conditions, with increased quenching upon addition of S1-ADP or Ca²⁺.

Figure 2

Ca²⁺ titration curves for cTnI(167C_AEDANS)-cTnC(89C_DDPM) or cTnI(151C_AEDANS)-cTnC(89C_DDPM) labeled thin filaments containing either wild type or mutant cTnT. The normalized FRET efficiency increases as AEDANS and DDPM are brought into closer proximity upon addition of gradually increasing levels of Ca²⁺.

Figure 3

Representative fluorescence decays for donor only and cTnI(S167C_AEDANS)-cTnC(S89C_DDPM) labeled thin filaments. Donor-acceptor decays are given for four physiological conditions; with Ca²⁺ or without Ca²⁺, and with or without S1-ADP. The donor only decay gives the longest lifetime followed by donor-acceptor (DA) without Ca²⁺. Addition of S1-ADP decreases the lifetime in the Ca²⁺ free state but not Ca²⁺ saturated conditions and addition of Ca²⁺ decreased the lifetime for all DA samples with or without S1-ADP. Donor only samples were unchanged for all conditions as was expected.

Figure 4

Representative distance distributions derived by GlobalCurve fitting of fluoresence intensity decay data. Traces are for cTnI(S151C_AEDANS)-cTnC(S89C_DDPM)-cTnT(WT) samples in low Ca²⁺ without S1-ADP (solid blue trace), in low Ca²⁺ with S1-ADP present (solid red trace) and both with and without S1-ADP present under saturating levels of Ca²⁺ (solid red and blue traces respectively).

Figure 5

Column graph showing switching distances for cTnT mutants with or without S1-ADP, shown in red and black respectively. The graph at left is labeled at cTnI(151) with AEDANS while the graph at right is labeled at cTnI(167), acceptor labeling at cTnC(89C) for both. This graph shows Ca²⁺ induced proximity changes between cTnI(151) and cTnC(89) or cTnI(167) and cTnC(89). As can be seen switching distance is greater for cTnI(167) than cTnI(151) and samples with S1-ADP all show decreased switching distances compared to the same samples without S1-ADP. The impact of cTnT pseudo-phosphorylation is seen as a decrease in switching distances, which is most clearly pronounced in cTnT(3M) and cTnT(4M). Interestingly cTnT pseudo-phosphorylation's impact is lost upon addition of S1-ADP for the cTnI(151) labeled samples.

Figure 6

Representative BAPTA chelation traces for cTnT mutants S199E or 4M and control. The graph on the left shows samples using cTnI(S151C_AEDANS)-cTnC(S89C_DDPM) FRET pairs, while the graph on the right shows samples using cTnI(S167C_AEDANS)-cTnC(S89C_DDPM) FRET pairs. Deactivation kinetics at cTnI(151) are not significantly impacted by pseudo-phosphorylation mutants while at cTnI(167) deactivation kinetics are decreased.