Open Collections

Troponin C regulation of length-dependent calcium-sensitivity in rabbit skinned psoas muscle fibre segments

University of British Columbia

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.

Thesis/Dissertation

application/pdf

2009-01-24

For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use.

http://hdl.handle.net/2429/3863

Anatomy, Cell Biology and Physiology

University of British Columbia

UBCV

DSpace