Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium

1. Measurements of the unloaded sliding speed of and isometric force exerte d on single thin filaments in in vitro motility assays were made to evaluat e the role of regulatory proteins in the control of unloaded thin filament sliding speed and isometric force production. 2. Regulated actin filaments were reconstituted from rabbit F-actin, native bovine cardiac tropomyosin (nTm), and either native bovine cardiac troponi n (nTn), troponin containing a TnC mutant, CBMII, in which the sole regulat ory site in cardiac TnC (site II) is inactivated (CBMII-Tn), or troponin co ntaining a point mutation in TnT (179N, where isoleucine at position 79 is replaced with asparagine) associated with familial hypertrophic cardiomyopa thy (PHC). 3. Addition of regulatory proteins to the thin filament increases both the unloaded sliding speed and the isometric force exerted by myosin heads on t he thin filaments. 4. Variation of thin filament activation by varying [Ca2+] or the fraction of CBMII/TnC bound to the thin filament at pCa 5, had little effect on the unloaded filament sliding speed until the fraction of the thin filament con taining calcium bound to TnC was less than 0.15. These results suggest that [Ca2+] primarily affects the number of attached and cycling crossbridges. 5. The presence of the FHC TnT mutant increased the thin filament sliding s peed but reduced the isometric force that heavy meromyosin exerted on regul ated thin filaments. These latter results, together with the increased slid ing speed and isometric force seen in the presence of regulatory proteins, suggest that thin filament regulatory proteins exert significant allosteric effects on the interaction of crossbridges with the thin filament.