期刊
JOURNAL OF PHYSIOLOGY-LONDON
卷 540, 期 2, 页码 485-497出版社
CAMBRIDGE UNIV PRESS
DOI: 10.1113/jphysiol.2001.013179
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资金
- NHLBI NIH HHS [HL52558, HL65497, R01 HL061683, HL61683, R01 HL065497] Funding Source: Medline
The role of cooperative interactions between individual structural regulatory units (SUs) of thin filaments (7 actin monomers: I tropomyosin: 1 troponin complex) on steady-state Ca2+-activated force was studied. Native troponin C (TnC) was extracted from single, de-membranated rabbit psoas fibres and replaced by mixtures of purified rabbit skeletal TnC (sTnC) and recombinant rabbit sTnC (D27A, D63A), which contains mutations that disrupt Ca2+ coordination at N-terminal sites I and II (xxsTnC). Control experiments in fibres indicated that, in the absence of Ca2+, both sTnC and xxsTnC bind with similar apparent affinity to sTnC-extracted thin filaments. Endogenous sTnC-extracted fibres reconstituted with 100% xxsTnC did not develop Ca2+-activated force. In fibres reconstituted with mixtures of sTnC and xxsTnC, maximal Ca2+-activated force increased in a greater than linear manner with the fraction of sTnC. This suggests that Ca2+ binding to functional Tn can spread activation beyond the seven actins of an SU into neighbouring units, and the data suggest that this functional unit (FU) size is up to 10-12 actins. As the number of FUs was decreased, Ca2+ sensitivity of force (pCa(50)) decreased proportionally. The slope of the force-pCa relation (the Hill coefficient, n(H)) also decreased when the reconstitution mixture contained < 50% sTnC. With 15% sTnC in the reconstitution mixture, n(H) was reduced to 1.7 +/- 0.2, compared with 3.8 +/- 0.1 in fibres reconstituted with 100% sTnC, indicating that most of the cooperative thin filament activation was eliminated. The results suggest that cooperative activation of skeletal muscle fibres occurs primarily through spread of activation to near-neighbour FUs along the thin filament (via head-to-tail tropomyosin interactions).
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