Tropomyosin R160A is crucial for TnI phosphorylation-induced effects on calcium sensitivity.

Abstract

The thin filament, consisting of the three-protein complex troponin (TnI, TnT, and TnC) and tropomyosin (Tpm) bound to filamentous actin, acts as the main transducer that relates sarcoplasmic calcium levels to the contraction-relaxation cycles of the myocardium. In the absence of calcium, TnI biases Tpm in a position on actin that blocks myosin binding whereas calcium binding to TnC causes a propagated conformational change in Tn/Tpm that relieves this inhibition. During heart failure and adrenergic stimulation, phosphorylation of the N-terminal region of troponin I (TnI) at residues S23 and S24, has been shown to play a role in heart muscle adaptation through a decrease in calcium sensitivity. Previous modeling studies performed in thin filament models have suggested that positively charged groups on Tpm, namely R160, could interact with the phosphorylated serine residues on TnI. Here we tested the hypothesis that interactions between Tpm-R160 and phosphorylated S23/24 participate in phosphorylation-induced effects on calcium sensitivity (Pavadai et al., 2022). Tpm mutated from wild-type R160 to alanine (Tpm-R160A) and phosphomimetic TnI affecting both serine 23 and 24 (S23/24D) were generated and expressed recombinantly. Using in vitro motility (IVM) assays, we quantified the effects of these mutations alone and in combination. Regulated IVM experiments using thin filaments reconstituted with phosphomimetic S23/24D-TnI and wild-type Tpm revealed a decrease in calcium sensitivity, showing that phosphorylation is successfully mimicked by mutating serine to an aspartic acid,consistent with previous studies on human cardiac tissue. However, when TnI-S23/24D is paired with mutant R160A-Tpm, the phosphorylation-induced changes in calcium sensitivity are diminished. These results are consistent with Tpm-R160 participating in the lusitropy observed when the N-terminal of TnI is phosphorylated.