Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment

Lee RS, Tikunova SB, Kline KP, Zot HG, Hasbun JE, Van Minh N, Swartz DR, Rall JA, Davis JP. Effect of Ca2+ binding properties of troponin C on rate of skeletal muscle force redevelopment. Am J Physiol Cell Physiol 299: C1091-C1099, 2010. First published August 11, 2010; doi:10.1152/ajpcell.00491.2009.-To investigate effects of altering troponin (Tn)C Ca2+ binding properties on rate of skeletal muscle contraction, we generated three mutant TnCs with increased or decreased Ca2+ sensitivities. Ca2+ 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 similar to 1.9-fold higher, similar to 5.0-fold lower, and similar to 52-fold lower Ca2+ sensitivity, respectively, and similar to 3.6-fold slower, similar to 5.7-fold faster, and similar to 21-fold faster Ca2+ dissociation rate (k(off)), respectively. On the basis of K-d and k(off), these results suggest that the Ca2+ association rate to the Tn complex decreased similar to 2-fold for I60QTnC and V43QTnC. Constructs were reconstituted into single-skinned rabbit psoas fibers to assess Ca2+ dependence of force development and rate of force redevelopment (k(tr)) at 15 degrees C, resulting in sensitization of both force and k(tr) to Ca2+ for V43QTnC, whereas T70DTnC and I60QTnC desensitized force and k(tr) to Ca2+, 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 Ca2+ 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 Ca2+ concentration. Thus TnC mutants with altered Ca2+ binding properties can control the rate of contraction by modulating thin filament activation without directly affecting intrinsic cross-bridge cycling rates.