Myofibrillar troponin exists in three states and there is signal transduction along skeletal myofibrillar thin filaments.
Activation of striated muscle contraction is a highly cooperative signal transduction process converting calcium binding by troponin C (TnC) into interactions between thin and thick filaments. Once calcium is bound, transduction involves changes in protein interactions along the thin filament. The process is thought to involve three different states of actin-tropomyosin (Tm) resulting from changes in troponin's (Tn) interaction with actin-Tm: a blocked (B) state preventing myosin interaction, a closed (C) state allowing weak myosin interactions and favored by calcium binding to Tn, and an open or M state allowing strong myosin interactions. This was tested by measuring the apparent rate of Tn dissociation from rigor skeletal myofibrils using labeled Tn exchange. The location and rate of exchange of Tn or its subunits were measured by high-resolution fluorescence microscopy and image analysis. Three different rates of Tn exchange were observed that were dependent on calcium concentration and strong cross-bridge binding that strongly support the three-state model. The rate of Tn dissociation in the non-overlap region was 200-fold faster at pCa 4 (C-state region) than at pCa 9 (B-state region). When Tn contained engineered TnC mutants with weakened regulatory TnI interactions, the apparent exchange rate at pCa 4 in the non-overlap region increased proportionately with TnI-TnC regulatory affinity. This suggests that the mechanism of calcium enhancement of the rate of Tn dissociation is by favoring a TnI-TnC interaction over a TnI-actin-Tm interaction. At pCa 9, the rate of Tn dissociation in the overlap region (M-state region) was 100-fold faster than the non-overlap region (B-state region) suggesting that strong cross-bridges increase the rate of Tn dissociation. At pCa 4, the rate of Tn dissociation was twofold faster in the non-overlap region (C-state region) than the overlap region (M-state region) that likely involved a strong cross-bridge influence on TnT's interaction with actin-Tm. At sub-maximal calcium (pCa 6.2-5.8), there was a long-range influence of the strong cross-bridge on Tn to enhance its dissociation rate, tens of nanometers from the strong cross-bridge. These observations suggest that the three different states of actin-Tm are associated with three different states of Tn. They also support a model in which strong cross-bridges shift the regulatory equilibrium from a TnI-actin-Tm interaction to a TnC-TnI interaction that likely enhances calcium binding by TnC.
Swartz
Darl R
DR
Indiana University School of Medicine, Department of Anatomy and Cell Biology, Indianapolis, IN 46202, USA. drswartz@purdue.edu
Yang
Zhenyun
Z
Sen
Asok
A
Tikunova
Svetlana B
SB
Davis
Jonathan P
JP
D000199:Actins / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002645:Chickens; D066298:In Vitro Techniques; D008954:Models, Biological; D009119:Muscle Contraction / Q000502:physiology; D018482:Muscle, Skeletal / Q000502:physiology*; D009210:Myofibrils / Q000502:physiology*; D011485:Protein Binding; D011817:Rabbits; D015398:Signal Transduction; D014335:Tropomyosin / Q000502:physiology; D019209:Troponin C / Q000502:physiology*; D019210:Troponin I / Q000502:physiology*; D020107:Troponin T / Q000502:physiology*
D000199:Actins; D014335:Tropomyosin; D019209:Troponin C; D019210:Troponin I; D020107:Troponin T; D002118:Calcium