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.