Abstract
Cardiac muscle thin filaments are composed of actin, tropomyosin, and troponin that change conformation in response to Ca2+ binding, triggering muscle contraction. Human cardiac troponin C (cTnC) is the Ca2+-sensing component of the thin filament. It contains structural sites (III/IV) that bind both Ca2+ and Mg2+ and a regulatory site (II) that has been thought to bind only Ca2+. Binding of Ca2+ at this site initiates a series of conformational changes that culminate in force production. However, the mechanisms that underpin the regulation of binding at site II remain unclear. Here, we have quantified the interaction between site II and Ca2+/Mg2+ through isothermal titration calorimetry and thermodynamic integration simulations. Direct and competitive binding titrations with WT N-terminal cTnC and full-length cTnC indicate that physiologically relevant concentrations of both Ca2+/Mg2+ interacted with the same locus. Moreover, the D67A/D73A N-terminal cTnC construct in which two coordinating residues within site II were removed was found to have significantly reduced affinity for both cations. In addition, 1 mM Mg2+ caused a 1.4-fold lower affinity for Ca2+. These experiments strongly suggest that cytosolic-free Mg2+ occupies a significant population of the available site II. Interaction of Mg2+ with site II of cTnC likely has important functional consequences for the heart both at baseline as well as in diseased states that decrease or increase the availability of Mg2+, such as secondary hyperparathyroidism or ischemia, respectively.
Highlights
Cardiac troponin is a heterotrimeric complex that includes components for Ca2+ binding, inhibition of contraction, and tropomyosin binding [1]
The ratio of the ligand to titrant in the single-binding site condition is a measure of the functional moles of protein and was approximately 1.00 in all the N-cardiac troponin C (cTnC) titrations (Table S2)
The values presented can be compared between conditions, but care should be taken when comparing these to other systems, such as the Cardiac troponin (cTn) complex or the reconstituted thin filaments [59, 60]
Summary
Cardiac troponin (cTn) is a heterotrimeric complex that includes components for Ca2+ binding (cardiac troponin C [cTnC]), inhibition of contraction (cardiac troponin I [cTnI]), and tropomyosin binding (cardiac troponin T [cTnT]) [1]. Given the abundance of contractile filaments throughout cardiomyocytes, sites III/IV of cTnC buffer 80% of the 100 to 200 μM [Ca2+]in at resting concentrations of free Ca2+ (100 nM) [16]. At resting free cytosolic Ca2+ concentrations, sites III and IV are usually saturated with Ca2+ [16]. The cytosolic concentration of Mg2+ allows this cation to compete with and reduce the binding of Ca2+ to the “structural” sites [17, 18]. The binding of Ca2+/Mg2+ to sites III and IV alters the structure of TnC and is a prerequisite for tethering to the rest of the TF [19, 20]
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