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 Ca_V1.3 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.