Kinetics of Thin Filament Activation Probed by Fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-Labeled Troponin I Incorporated into Skinned Fibers of Rabbit Psoas Muscle: Implications for Regulation of Muscle Contraction

AbstractMaking use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (Trybus and Taylor, 1980. Proc. Natl. Acad. Sci. 77:7209–7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (Brenner et al., 1999. Biophys. J. 77:000–000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca2+ concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (