The role of the C-domain sites of cardiac
troponin C in the modulation of the
calcium signal remains unclear. In this study, we investigated the effects of
hypertrophic cardiomyopathy-linked mutations A8V, E134D, and D145E in cardiac
troponin C on the properties of the C-domain sites. The A8V mutation had essentially no effect on the
calcium or
magnesium binding properties of the C-domain sites, while the mutation E134D moderately decreased
calcium and
magnesium binding affinities. On the other hand, the D145E mutation affected cooperative interactions between sites III and IV, significantly reducing the
calcium binding affinity of both sites. Binding of the anchoring region of cardiac
troponin I (corresponding to residues 34-71) to cardiac
troponin C with the D145E mutation was not able to recover normal
calcium binding to the C-domain. Experiments utilizing the fluorescent hydrophobic probe
bis-ANS suggest that the D145E mutation dramatically reduced the extent of
calcium-induced hydrophobic exposure by the C-domain. At high nonphysiological
calcium concentration, A8V, E134D, and D145E mutations minimally affected the affinity of cardiac
troponin C for the regulatory region of cardiac
troponin I (corresponding to residues 128-180). In contrast, at lower physiological
calcium concentration, the D145E mutation led to an approximately 8-fold decrease in the affinity of cardiac
troponin C for the regulatory region of cardiac
troponin I. Our results suggest that
calcium binding properties of the C-domain sites might be important for the proper regulatory function of cardiac
troponin C.