Although the sliding filament mechanism is generally accepted as the way in which actin and myosin interact in order to produce movement, controversy still surrounds the detailed molecular interactions of these proteins in intact fiber systems. Although much is known about the role of Ca2+ binding to Troponin (Tn) in the regulation of cardiac and skeletal muscle contraction, it is based primarily upon studies in solution of the various proteins involved and these results have been simply extrapolated to the intact muscle fiber. Recent evidence suggests that the Ca2+ binding properties of the Ca2+-specific sites of Tn are altered when Tn is incorporated into the thin filament and of equal importance, other studies suggest that myosin crossbridge interaction with the actin filament during contraction may also affect Ca2+ binding. Until recently it has not been possible to address these questions directly. Recent advances now make it possible to study these as well as other related phenomena in systems of increasing complexity and structural integrity, including thin filaments, myofibrils, skinned fibers and intact muscle fibers. These experiments became possible through the discovery that troponin C (TnC), the Ca2+ binding subunit of Tn can be selectively extracted from myofibrils or skinned fibers, inhibiting either Ca2+ dependent myofibrillar ATPase or tension development, respectively. It was found that both activities can be fully restored upon readdition of either native cardiac (C) or skeletal (S) TnC or fluorescently modified derivatives of these proteins. Since these fluorescent proteins undergo well characterized changes in fluorescence upon binding Ca2+ at the various classes of Ca2+ binding sites on TnC (Ca2+-Mg2+ or Ca2+-specific), it is possible using these techniques to measure the Ca2+ affinity of these sites in reconstituted myofibrils and skinned fibers in the steady state and as a function of time. Using microspectro-fluorometry we have been able to measure Ca2+ affinity, tension development and ATPase activity in skinned fibers. We propose here to use these procedures to determine the Ca2+ affinity of the Ca2+ binding sites on Tn in these reconstituted systems and how they are affected in relation to the other parameters by a variety of modifiers (e.g. crossbridge attachment, rigor, pH, Mg2+, MgATP2-, cardiac TnI phosphorylation, etc.). Mechanical manipulations of the skinned fibers will be used as well as caged ATP to perturb the crossbridge state and probe the Ca2+ affinity of these sites. Direct injection of fluorescent TnC* derivatives into intact Balanus nubilus fibers has been useful in estimating the extent of filament bound Ca2+ in relation to isometric force. In this case TnC* is not associated with filaments yet has the same Ca2+ affinity as the TnC in Tn on the thin filament. Research will be further developed and extended to single frog skeletal muscle fibers. Time resolved X-ray diffraction studies will be employed to correlate the time course of crossbridge attachment and tropomyosin movement with simultaneously measured filament bound Ca2+, and free Ca2+ using the techniques described above in the skinned as well as in the intact fiber. The combined results should yield a much clearer view of the temporal and molecular events involved with muscle activation.
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