title: Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment.
abstract: To investigate effects of altering troponin (Tn)C Ca(2+) binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca(2+) sensitivities. Ca(2+) binding properties of the regulatory domain of TnC within the Tn complex were characterized by following the fluorescence of an IAANS probe attached onto the endogenous Cys(99) residue of TnC. Compared with IAANS-labeled wild-type Tn complex, V43QTnC, T70DTnC, and I60QTnC exhibited ∼1.9-fold higher, ∼5.0-fold lower, and ∼52-fold lower Ca(2+) sensitivity, respectively, and ∼3.6-fold slower, ∼5.7-fold faster, and ∼21-fold faster Ca(2+) dissociation rate (k(off)), respectively. On the basis of K(d) and k(off), these results suggest that the Ca(2+) association rate to the Tn complex decreased ∼2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca(2+) dependence of force development and rate of force redevelopment (k(tr)) at 15°C, resulting in sensitization of both force and k(tr) to Ca(2+) for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca(2+), I60QTnC causing a greater desensitization. In addition, T70DTnC and I60QTnC depressed both maximal force (F(max)) and maximal k(tr). Although V43QTnC and I60QTnC had drastically different effects on Ca(2+) binding properties of TnC, they both exhibited decreases in cooperativity of force production and elevated k(tr) at force levels <30%F(max) vs. wild-type TnC. However, at matched force levels >30%F(max) k(tr) was similar for all TnC constructs. These results suggest that the TnC mutants primarily affected k(tr) through modulating the level of thin filament activation and not by altering intrinsic cross-bridge cycling properties. To corroborate this, NEM-S1, a non-force-generating cross-bridge analog that activates the thin filament, fully recovered maximal k(tr) for I60QTnC at low Ca(2+) concentration. Thus TnC mutants with altered Ca(2+) binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.
authors: Department of Physiology and Cell Biology, Ohio State University, Columbus, Ohio, USA.
authors: Ryan S
authors: RS
authors: Lee
authors: Svetlana B
authors: SB
authors: Tikunova
authors: Kristopher P
authors: KP
authors: Kline
authors: Henry G
authors: HG
authors: Zot
authors: Javier E
authors: JE
authors: Hasbun
authors: Nguyen Van
authors: NV
authors: Minh
authors: Darl R
authors: DR
authors: Swartz
authors: Jack A
authors: JA
authors: Rall
authors: Jonathan P
authors: JP
authors: Davis
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology*; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011817:Rabbits; D014336:Troponin / Q000378:metabolism; D019209:Troponin C / Q000235:genetics / Q000378:metabolism*
chemical_list: D014336:Troponin; D019209:Troponin C; D002118:Calcium