Effects of Cardiac Troponin C Mutants on TnC-TnI interaction and its modulation by PKA phosphorylation

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We are considering several cardiac TnC mutants as potential therapeutic strategies for cardiomyopathies. To judge their potential to improve in situ function it is important to understand how these mutants affect TnC-TnI interaction and its modulation by PKA-mediated phosphorylation of TnI Ser 23, Ser 24. In this study, we are characterizing two cardiac TnC mutations, Leu48Glu (L48Q) and Ile61Glu (I61Q), with increased and decreased (respectively) Ca2+ binding affinity. In previous studies we showed these mutations resulted in increased (L48Q) and decreased (I61Q) Ca2+ sensitivity of steady state force in skinned rat trabeculae. To determine if these changes in Ca2+ sensitivity were due to altered TnC-TnI interactions we generated a structural marker by attaching IANBD to Cys84 in the N-lobe of cTnC. Half-maximal IANBD fluorescence saturation of Ca2+ binding occurred at pCa7.42 for L48Q cTnC, 7.38 for wild-type (WT) cTnC and 7.30 for I61Q cTnC. In both the absence and presence of saturating Ca2+ (0.6 μM TnC) IANBD fluorescence increased with increasing TnI and saturated at different [TnI] in the order L48Q, WT, I61Q. Fluorescence half-maximal saturation occurred at 0.26μM(saturating Ca2+) and 0.25 μM (no Ca2+)TnI for L48Q cTnC, 0.78 μM and 0.49μM for WT cTnC , and 1.45 μM and 0.69 μM I61Q cTnC according to the exponential function fit of the data. However, preliminary experiments suggest that when PKA phosphorylated [cTnI] was titrated to cTnC, in both the absence and presence of saturating Ca2+, IANBD fluorescence enhancement with L48Q may be impaired. The data thus far suggest that single amino acid mutations that alter Ca2+ binding affinity of TnC can influence interaction with TnI and its modulation by PKA mediated phosphorylation of Ser 23, Ser 24. Supported by HL65497 to MR.