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

Cardiac troponin C (cTnC) is the calcium (Ca 2+ ) sensing component of the troponin complex. Binding of Ca 2+ to cTnC triggers a cascade of myofilament conformational changes that culminate in force production. Mutations in cTnC linked to hypertrophic myocardial myopathy (HCM) induce a a greater degree and duration of Ca 2+ binding, which may underly the hypertrophic phenotype. Recent evidence from our laboratories demonstrated novel modifications of cTnC Ca 2+ binding by cellular magnesium (Mg 2+ ) that we hypothesize may be of significance in promoting HCM. Regulation of contraction has long been thought to occur exclusively through Ca 2+ binding to site II of cTnC. However, abundant cellular Mg 2+ is a potential competitor for binding to the same sites; work by several groups also suggests this is possible. We have used isothermal titration calorimetry (ITC) to explore the thermodynamic properties associated with the interaction between Ca 2+ /Mg 2+ and site II of cTnC; these experiments demonstrated that physiological concentrations of Mg 2+ may compete with Ca 2+ to bind site II of cTnC. In experiments reported here, we studied a series of mutations in cTnC thought to be causal in HCM. Three mutants (A8V, L29Q, and A31S) slightly elevated the affinity for both Ca 2+ and Mg 2+ , whereas other mutants (L48Q, Q50R, and C84Y), that are closer to the C-terminal domain and surrounding the EF hand binding motif of site II had a more significant effect on affinity and the thermodynamics of the binding interaction. To the best of our knowledge, this work is the first to explore the role of Mg 2+ in modifying the Ca 2+ affinity ofcTnC mutations linked to HCM. Our results indicate a physiologically significant role for cellular Mg 2+ at baseline conditions and when elevated on the control of the dynamics of contraction by modifications in the Ca 2+ binding properties of cTnC.