Simultaneous detection of reciprocal interactions between calmodulin, Ca2+ and molecular targets: a focus on the calmodulin-RyR2 complex

In a recent issue of Biochemical Journal, Brohus et al. (Biochem. J.476, 193-209) investigated the interaction between the ubiquitous intracellular Ca2+-sensor calmodulin (CaM) and peptides that mimic different structural regions of the cardiac ryanodine receptor (RyR2) at different Ca2+ concentrations. For the purpose, a novel bidimensional titration assay based on changes in fluorescence anisotropy was designed. The study identified the CaM domains that selectively bind to a specific CaM-binding domain in RyR2 and demonstrated that the interaction occurs essentially under Ca2+-saturating conditions. This study provides an elegant and experimentally accessible framework for detailed molecular investigations of the emerging life-threatening arrhythmia diseases associated with mutations in the genes encoding CaM. Furthermore, by allowing the measurement of the equilibrium dissociation constant in a protein-protein complex as a function of [Ca2+], the methodology presented by Brohus et al. may have broad applicability to the study of Ca2+ signalling.