Protein Kinase C Phosphomimetics Alter Thin Filament Ca2+ Binding Properties

Adrenergic stimulation modulates cardiac function by altering the phosphorylation status of several cardiac proteins. The Troponin complex, which is the Ca2+ sensor for cardiac contraction, is a hot spot for adrenergic phosphorylation. While the effect of beta-adrenergic related PKA phosphorylation of troponin I at Ser23/24 is well established, the effects of alpha-adrenergic induced PKC phosphorylation on multiple sites of TnI (Ser43/45, Thr144) and TnT (Thr194, Ser198, Thr203 and Thr284) are much less clear. By utilizing an IAANS labeled fluorescent troponin C, TnC(IAANS)(T53C), we systematically examined the site specific effects of PKC phosphomimetic mutants of TnI and TnT on TnC's Ca2+ binding properties in the Tn complex and reconstituted thin filament. The majority of the phosphomemetics had little effect on the Ca2+ binding properties of the isolated Tn complex. However, when incorporated into the thin filament, the phosphomimetics typically altered thin filament Ca2+ sensitivity in a way consistent with their respective effects on Ca2+ sensitivity of skinned muscle preparations. The altered Ca2+ sensitivity could be generally explained by a change in Ca2+ dissociation rates. Within TnI, phosphomimetic Asp and Glu did not always behave similar, nor were Ala mutations (used to mimic non-phosphorylatable states) benign to Ca2+ binding. Our results suggest that Troponin may act as a hub on the thin filament, sensing physiological stimuli to modulate the contractile performance of the heart.