Modulations of CA2+ Sensitizers and Phosphorylation of cTnT in Dynamic Equilibrium of cTnC N-Domain

Abstract

An increasing body of evidence suggests cTnC exists in dynamic conformational equilibrium and that Ca2+ binding regulates this equilibrium. However, little direct evidence has been offered to support these claims. The present study aims to directly monitor dynamic conformational equilibrium of cTnC and investigate how the dynamic equilibrium is modulated by the presence of cardiac troponin I (cTnI), cardiac troponin T (cTnT), Ca2+ sensitizers, and a Ca2+ desensitizing phosphomimic of cTnT (cTnT(T204E). To achieve the objective, a dye homodimerization approach is developed and implemented which allows for determination of the dynamic equilibrium between open and closed conformations in cTnC's hydrophobic cleft. Modulation of this equilibrium by other troponin subunits, Ca2+-sensitizers, and phosphomimic of cTnT(T204E) is characterized. Isolated cTnC contained a small open conformation population in the absence of Ca2+, which increased significantly upon addition of saturating levels of Ca2+. This suggests the Ca2+ induced activation of thin filament arises from an increase in the probability of hydrophobic cleft opening. Inclusion of cTnI increased the population of open cTnC while inclusion of cTnT had the opposite effect. Samples containing Ca2+-desensitizing cTnT(T204E) showed a slight but insignificant decrease in open conformation probability compared to samples with cTnT(wt), while Ca2+-sensitizer treated samples generally increased open conformation probability. These findings show that an equilibrium between open and closed conformations of cTnC's hydrophobic cleft are play a significant role in tuning the Ca2+ sensitivity of the heart.