| Summary: | The involvement of troponin C (TnC) in regulating the length dependence of
calcium sensitivity was studied in rabbit skinned psoas muscle fibre segments. Force-pCa
curves were acquired from these isolated segments at resting sarcomere lengths (2.4 µm)
and at lengths (3.0 µm) longer than the plateau of the tension-length relationship. Partial
extraction of endogenous TnC from fibre segments was performed by bathing the fibre in
a solution consisting of 20 mM imidazole and 5 mM ethylenediaminetetraacetate at pH
7.85, while a more complete removal of TnC was facilitated by the addition of 1 mM
trifluoperazine to the extraction medium. Treated fibres were then reconstituted with
either native rabbit skeletal TnC or a mutant TnC in which the amino acid residue at
position 130 of the protein's high-affinity domain was replaced with serine (I130S), glycine
(I130G) or recombinant isoleucine (1130). These TnC mutants have been shown
previously to possess destabilized alpha-helices in the protein's carboxy-terminal domain
resulting in altered ion-affinities of the associated Ca²⁺/Mg²⁺ binding sites (Trigo-Gonzalez et al, 1993). Protein removal and replacement were assayed by examining
changes in the fibre's ability to generate contractile force and subsequently confirmed by
silver stained sodium dodecylsulfate polyacrylamide gels of fibre segments. Following
fibre reconstitution, force-pCa relations were measured again at both resting and long
sarcomere lengths. Results from this study indicate that myofilament calcium sensitivity is
neither affected by the method of endogenous TnC extraction nor by mutations to this
region of TnC's high-affinity domain. === Medicine, Faculty of === Graduate
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