Interaction of bepridil with the cardiac troponin C/troponin I complex

We have investigated the binding of bepridil to calcium-saturated cardiac t roponin C in a cardiac troponin C/troponin I complex. Nuclear magnetic reso nance spectroscopy and [N-15,H-2]cardiac troponin C permitted the mapping o f bepridil-induced amide proton chemical shifts. A single bepridil-binding site in the regulatory domain was found with an affinity constant of simila r to 140 muM(-1). In the presence of cardiac troponin I, bepridil binding t o the C domain of cardiac troponin C was not detected. The pattern of bepri dil-induced chemical shifts is consistent with stabilization of more open r egulatory domain conformational states. A similar pattern of chemical shift perturbations was observed for interaction of the troponin I cardiac-speci fic amino-terminus with the cardiac troponin C regulatory domain. These res ults suggest that both bepridil and the cardiac-specific amino-terminus may mediate an increase in calcium affinity by interacting with and stabilizin g open regulatory domain conformations. Chemical shift mapping suggests a p ossible role for inactive calcium-binding site I in the modulation of calci um affinity. (C) 2001 Federation of European Biochemical Societies. Publish ed by Elsevier Science B.V. All rights reserved.