• Potter, James D. (PI)

Project: Research project

Project Details


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.
Effective start/end date7/1/868/31/97


  • National Institutes of Health
  • National Institutes of Health: $307,605.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $297,691.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health


  • Medicine(all)

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