WHAT DOES TNCDANZ FLUORESCENCE REVEAL ABOUT THE THIN FILAMENT STATE

Fluorescence of skinned psoas fibres reconstituted with the troponin C subunit labelled with the fluorescent probe dansylaziridine (TnC(DANZ)) increases upon activation with Ca2+. This fluorescence enhancement is due to Ca2+ binding to the Ca2+-specific binding sites of TnC(DANZ) and attachment of cross-bridges to the actin filament. We found that approximately 20% of the enhanced fluorescence signal derived from Ca2+ binding to TnC(DANZ) and 80% from cross-bridge attachment during maximal activation. Furthermore we studied the effects of different cross-bridge states on TnC(DANZ) fluorescence. "Weakly" bound, non-force-generating cross-bridge states (pCa 8, low ionic strength) and rigor cross-bridges revealed similar effects on the TnC(DANZ) fluorescence. "Strongly" attached, force-generating states, however, increased fluorescence to the greatest extent. These results suggests a complex system of reciprocal couplings between TnC and different attached cross-bridge states. Cooling or increase of inorganic phosphate decreased isometric force but hardly decreased fluorescence, suggesting the accumulation of attached cross-bridge states with low tension output.