Molecular basis for a Ca2+ binding hierarchy in the EF-hand motifs of CaBP2.

Calcium binding protein 2 (CaBP2), a member of the calmodulin (CaM) superfamily, modulates the L-type calcium channel CaV1.3 in the cochlear inner hair cells by suppressing its voltage- and Ca2+-dependent inactivation. Like CaM, CaBP2 contains four EF-hand motifs, although only three are functional, yet the precise nature and physiological relevance of its Ca2+ binding remain poorly understood. In this study, we present the binding properties of the individual EF-hand motifs of CaBP2L for Ca2+ and Mg2+ using isothermal titration calorimetry, and nuclear magnetic resonance spectroscopy. The measured dissociation constants of the individual EF-hand motifs span several orders of magnitude, suggesting a hierarchical mode of Ca2+ sensing. Our data demonstrate that the CaBP2L structure is stabilized upon Ca2+ rather than Mg2+ binding. While the N-lobe of CaBP2L significantly contributes to structural fluctuation in the absence of Ca2+, this fluctuation is reduced upon Ca2+ binding. Furthermore, our electrophysiology experiments show that upon disruption of all three Ca2+ binding EF hands of CaBP2 its ability to attenuate Ca2+-dependent inactivation is reduced. Therefore, we propose that CaBP2L displays a distinct Ca2+ binding property compared to other CaBPs, and that Ca2+ binding influences the stability of its structure and consequently channel regulation.