Mutations of hydrophobic residues in the n-terminal domain of troponin C affect calcium binding and exchange with the troponin C-troponin I96-148 complex and muscle force production

Interactions between troponin C and troponin I play a critical role in the regulation of skeletal muscle contraction and relaxation. We individually substituted 27 hydrophobic Phe, Ile, Leu, Val, and Met residues in the regulatory domain of the fluorescent troponin C-F29W with polar Gln to examine the effects of these mutations on: (a) the calcium binding and dynamics of troponin CF29W complexed with the regulatory fragment of troponin I (troponin I96-148) and (b) the calcium sensitivity of force production. Troponin I96-148 was an accurate mimic of intact troponin I for measuring the calcium dynamics of the troponin C-F29W-troponin I complexes. The calcium affinities of the troponin C-F29W-troponin I96-148 complexes varied similar to243-fold, whereas the calcium association and dissociation rates varied similar to38- and similar to33-fold, respectively. Interestingly, the effect of the mutations on the calcium sensitivity of force development could be better predicted from the calcium affinities of the troponin C-F29W-troponin I96-148 complexes than from that of the isolated troponin C-F29W mutants. Most of the mutations did not dramatically affect the affinity of calcium-saturated troponin C-F29W for troponin I96-148. However, the Phe(26) to Gln and Ile(62) to Gln mutations led to > 10-fold lower affinity of calcium-saturated troponin C-F29W for troponin I96-148, causing a drastic reduction in force recovery, even though these troponin C-F29W mutants still bound to the thin filaments. In conclusion, elucidating the determinants of calcium binding and exchange with troponin C in the presence of troponin I provides a deeper understanding of how troponin C controls signal transduction.