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Influence of enhanced troponin C Ca 2+ ‐binding affinity on cooperative thin filament activation in rabbit skeletal muscle
Author(s) -
Kreutziger Kareen L.,
Gillis Todd E.,
Davis Jonathan P.,
Tikunova Svetlana B.,
Regnier Michael
Publication year - 2007
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2007.135426
Subject(s) - chemistry , biophysics , biology
We studied how enhanced skeletal troponin C (sTnC) Ca 2+ ‐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 Ca 2+ 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 Ca 2+ sensitivity of steady‐state force (pCa 50 ) 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 Ca 2+ at N‐terminal sites. Reconstitution with sTnC: xxsTnC mixtures to 20% of pre‐exchanged maximal force reduced pCa 50 by 0.35 for sTnC: xxsTnC, 0.25 for M80Q sTnC: xxsTnC, and 0.10 for M80Q sTnC F27W : xxsTnC. It is interesting that pCa 50 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 Ca 2+ ‐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 Ca 2+ affinity strongly influences Ca 2+ sensitivity of steady‐state force without affecting near‐neighbour thin filament RU cooperative activation or the relative contribution of crossbridges versus Ca 2+ to thin filament activation.