Reciprocal coupling between troponin C and myosin crossbridge attachment.
The attachment of cycling myosin crossbridges to actin and the resultant muscle contraction are regulated in skeletal muscle by the binding of Ca2+ to the amino-terminal, regulatory sites of the troponin C (TnC) subunit of the thin filament protein troponin. Conversely, the attachment of crossbridges to actin has been shown to alter the affinity of TnC for Ca2+. In this study, fluorescently labeled TnC incorporated into reconstituted thin filaments was used to investigate the relationship between crossbridge attachment to actin and structural changes in the amino-terminal region of TnC. Fluorescence intensity changes were measured under the following conditions: saturating [Ca2+] in the absence of crossbridges, rigor crossbridge attachment in the presence and absence of Ca2+, and cycling crossbridge attachment. The percent of heavy meromyosin crossbridges associated with the thin filaments under these conditions was also determined. The results show that, in addition to the binding of Ca2+ to TnC, the attachment of both rigor and cycling crossbridges to actin alters the structure of TnC near the regulatory, Ca2+-specific sites of the molecule. A differential coupling between weakly versus strongly bound crossbridge states and TnC structure was detected, suggesting a possible differential regulation of these states by conformational changes in TnC. These findings illustrate a reciprocal coupling, via thin filament protein interactions, between structural changes in TnC and the attachment of myosin crossbridges to actin, such that each can influence the other, and indicate that TnC is not simply an on-off switch but may exist in a number of different conformations.
Zot
A S
AS
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Potter
J D
JD
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D066298:In Vitro Techniques; D009119:Muscle Contraction; D009132:Muscles / Q000378:metabolism; D009218:Myosins / Q000378:metabolism*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014336:Troponin / Q000378:metabolism*; D019209:Troponin C
D000199:Actins; D014336:Troponin; D019209:Troponin C; D009218:Myosins; D002118:Calcium