Effect of 3-Cl-DPA on the Ca²⁺ binding properties of the regulatory N-domain of cTnC reconstituted into the cTn complex. (A) Decreases in IAANS fluorescence occur when Ca²⁺ binds to labeled cTnC with C35S, T53C, and C84S substitutions, reconstituted into the cTn complex in the absence or presence of increasing concentrations of 3-Cl-DPA or bepridil. Data sets were individually normalized and fit with a logistic sigmoid. (B) Plot of the apparent rates of Ca²⁺ dissociation from the cTn complex in the presence of increased concentrations of 3-Cl-DPA, TFP, or bepridil. (C) Representative stopped-flow traces as Ca²⁺ is removed from the cTn complex in the absence or presence of 100 µM 3-Cl-DPA, TFP, or bepridil. The traces have been normalized and staggered for clarity. Rates of Ca²⁺ dissociation were measured following the fluorescence of IAANS attached to Cys⁵³ of cTnC with C35S, T53C, and C84S substitutions after reconstitution into the cTn complex.