Key result
Single-cysteine substitutions in site II of cardiac troponin C showed that the threonine mutation reduced Ca2+ sensitivity while the phenylalanine mutation increased it in myofilaments.
Specific non-coordinating residues in site II of cardiac troponin C play crucial roles in determining the calcium sensitivity of force development in myofilaments.
Animal troponin C findings require human validation before any therapeutic consideration; leaves open site II residue roles in disease models.
Myocardial contraction is initiated when Ca2+ binds to site II of cardiac troponin C. This 12-residue EF-hand loop (NH2-DEDGSGTVDFDE-COOH) contains six residues (bold) that coordinate Ca2+ binding and six residues that do not appear to influence Ca2+ binding directly. We have introduced six single-cysteine substitutions (italics) within site II of cTnC to investigate whether these residues are essential for Ca2+ binding affinity in isolation and Ca2+ sensitivity of force development in single muscle fibers. Ca2+ binding properties of mutant proteins were examined in solution and after substitution into rat skinned soleus fibers. Except for the serine mutation, cysteine substitution had no effect on Ca2+ binding on cTnC in solution. However, as part of the myofilament, the threonine mutation reduced Ca2+ sensitivity while the phenylalanine mutation increased Ca2+ sensitivity. Analysis of the available crystal and NMR structures reveals specific structural mechanisms for these effects.
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Reece et al. (2008) studied this question. Single-cysteine substitutions within site II of cTnC was evaluated on Ca2+ binding affinity and Ca2+ sensitivity of force development. Single-cysteine substitutions in site II of cardiac troponin C showed that the threonine mutation reduced Ca2+ sensitivity while the phenylalanine mutation increased it in myofilaments.
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