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This Article Full Text Full Text (PDF) All Versions of this Article: 583/1/337    most recent jphysiol.2007.135426v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kreutziger, K. L. Articles by Regnier, M. Search for Related Content PubMed PubMed Citation Articles by Kreutziger, K. L. Articles by Regnier, M. Related Collections Skeletal Muscle and Exercise

¹ Department of Bioengineering, University of Washington, Seattle, WA 98195, USA
² Department of Physiology and Cell Biology, The Ohio State University, Columbus, OH, USA

We studied how enhanced skeletal troponin C (sTnC) Ca²⁺-binding affinity affects cooperative thin filament activation and contraction in single demembranated rabbit psoas fibres. Three sTnC mutants were created and incorporated into skeletal troponin (sTn) for measurement of Ca²⁺ dissociation, resulting in the following order of rates: wild-type (WT) sTnC–sTn > sTnC^F27W–sTn > M80Q sTnC–sTn > M80Q sTnC^F27W–sTn. Reconstitution of sTnC-extracted fibres increased Ca²⁺ sensitivity of steady-state force (pCa₅₀) by 0.08 for M80Q sTnC, 0.15 for sTnC^F27W and 0.32 for M80Q sTnC^F27W with minimal loss of slope (n_H, degree of cooperativity). Near-neighbour thin filament regulatory unit (RU) interactions were reduced in fibres by incorporating mixtures of WT or mutant sTnC and D28A, D64A sTnC (xxsTnC) that does not bind Ca²⁺ at N-terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre-exchanged maximal force reduced pCa₅₀ by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnC^F27W: xxsTnC. It is interesting that pCa₅₀ increased by 0.1 for M80Q sTnC and 0.3 for M80Q sTnC^F27W when near-neighbour RU interactions were reduced; these values are similar in magnitude to those for fibres reconstituted with 100% mutant sTnC. After reconstitution with sTnC: xxsTnC mixtures, n_H decreased to a similar value for all mutant sTnCs. Altered sTnC Ca²⁺-binding properties (M80Q sTnC^F27W) did not affect strong crossbridge inhibition by 2,3-butanedione monoxime when near-neighbour thin filament RU interactions were reduced. Together these results suggest increased sTnC Ca²⁺ affinity strongly influences Ca²⁺ sensitivity of steady-state force without affecting near-neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca²⁺ to thin filament activation.

(Received 26 April 2007; accepted after revision 13 June 2007; first published online 21 June 2007)
Corresponding author Michael Regnier: Department of Bioengineering, Box 355061, University of Washington, Seattle, WA 98195, USA. Email: mregnier{at}u.washington.edu

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