Binding of Calcium and Magnesium to Cardiac Troponin C

Abstract

Cardiac troponin C (cTnC) is the Ca 2+ -sensing component of the thin filament. It contains structural sites (III/IV) which bind both Ca 2+ and Mg 2+ , and a regulatory site (II) that has been thought to bind only Ca 2+ . The latter binding initiates a series of conformational changes that culminate in force production.We have quantified the interaction between site II and Ca 2+ /Mg 2+ through Isothermal Titration Calorimetry and Thermodynamic Integration simulations. Direct and competitive binding titrations using wild type and a double mutant that significantly reduces binding to site II demonstrated that physiologically relevant concentrations of both Ca 2+ /Mg 2+ interact with the same locus. Cytosolic free Mg 2+ (~1 mM) could occupy a significant population of available site II, as this concentration of Mg 2+ decreased the affinity for Ca 2+ 1.4-fold.Interaction of Mg 2+ with site II of cTnC likely has important functional consequences for the heart at baseline and in diseased states which decrease or increase availability of Mg 2+ such as secondary hyperparathyroidism or ischemia, respectively.

The effect of magnesium on calcium binding to cardiac troponin C related hypertrophic cardiomyopathy mutants

“…(71). Our recently published ITC and thermodynamic integration simulations corroborated these findings (57).…”

“…Previous ITC and thermodynamic integration (TI) simulations corroborate these findings; Mg 2+ competes with Ca 2+ for binding to site II and at physiological concentrations (1 mM) significantly reduces Ca 2+binding in full-length and N-terminal cTnC (57). In order to obtain a more complete picture of the cardiomyopathy mutations, it is important to establish whether they affect binding of Ca 2+ , Mg 2+ , or both.…”

“…al. Free energy (∆ ) corrections were made due to the introduction of the distance restraints and to correct for the charge of the system as described previously (57). For each system, 5 independent runs were performed and the results averaged.…”

“…A polar serine at residue 69 and a negatively charged glutamic acid at residue 76 in the EF hand binding site II of N-cTnC, create a domain that is conducive to Mg 2+ binding(87,88). We previously established the binding of Mg 2+ to site II in full length and N-terminal cTnC and would compete with Ca 2+ for binding to this site at physiological concentrations of each cation in the cell(57). In this work, we explore the hypothesis that Ca 2+ and Mg 2+ compete for binding, where affinity for each cation is allosterically modified by single amino acid changes outside the binding domain.Tikunova and Davis have shown that Mg 2+ , unlike Ca 2+ , does not cause a structural change upon binding but does significantly alter the affinity of cTnC for Ca 2+ with 3 mM Mg 2+ causing a more than 3-fold reduction in Ca 2+ binding affinity.…”

“…We measured a 47-fold difference in the affinity of WT N-cTnC for Ca 2+ (15.79 ± 0.74 µM) in comparison to Mg 2+ (711.08 ± 32.67 µM). However, Mg 2+ is at least 1000 times more abundant in the cytosol at systole than Ca 2+ 1 µM vs. 1 mM (89,90) and may compete for binding to site II in addition to the structural sites III and IV(57). Mg 2+ deficiency has been linked to cardiac disease including arrhythmias, hypertension, and congestive heart failure(91)(92)(93)(94).…”