H+ is involved in the activation of calcineurin by calmodulin

We have studied recently the surface hydrophobic properties of several calmodulin (CaM) target enzymes and found that a certain amphipathic domain(s) of the enzyme was exposed in response to a H+ increase within the neutral pH range. The exposed domain appeared to be related to their CaM-binding domains and associated with their activation by CaM (Huang, S., Carlson, G. M., and Cheung, W. Y. (1994) J. Biol. Chem. 269, 7631-7638). In this paper, the involvement of H+ in the activation of calcineurin (CaN), one of the CaM target enzymes, was further studied. Using dansylated CaM to monitor its conformational change, we found that the binding of Ca2+ to CaM occurred at a lower range of Ca2+ concentrations (pCa from 7 to 6) than the formation of CaM.CaN complex (pCa from 6 to 5). However, addition of H+ within the neutral pH range shifted the formation of CaM.CaN complex to the lower range of Ca2+ concentrations. Similarly, the addition of H+ shifted the Ca2+ requirement for enzyme activation to a lower Ca2+ range. These results show that the interaction of CaM with CaN could be initiated either by Ca2+ or by H+ in the presence of suboptimal Ca2+, suggesting a positive cooperative effect between Ca2+ and H+. The CaM-stimulated activity of CaN was accompanied by a time-dependent autoinactivation. Kinetic analysis showed that the autoinactivation initiated by Ca2+ or by H+ was biphasic and that the decay rate constants were comparable. In heavy water (D2O), the Ca(2+)-induced formation of CaM.CaN complex as well as the activity of CaN were decreased, indicating a solvent deuterium isotope effect. The same solvent isotope effect was observed in the H(+)-induced formation of CaM.CaN complex and in the H(+)-induced enzyme activity. When the enzyme was rendered CaM-independent by limited proteolysis, the isotope effect was abolished, suggesting that H+ was involved in the CaM-dependent and not the CaM-independent activity. Collectively, these data suggest that the interaction of CaM with CaN depends on the two cations: Ca2+ exposes an amphipathic domain on CaM, and H+ exposes the CaM-binding domain on the target enzyme.