High-throughput reevaluation of metal ion specificity in the Ca²⁺-binding photoprotein aequorin using automated...

Aequorin is a Ca²⁺-binding photoprotein that emits blue light upon binding calcium ions and has been widely used as a calcium indicator. Although the metal ion specificity of aequorin was investigated in the early 1970s, discrepancies among previous studies have left its ion selectivity incompletely understood. In this study, we systematically reevaluated the metal ion specificity of recombinant aequorin using an FDSS-μCELL® v2 automated imaging system equipped with integrated dispensing and highly sensitive luminescence detection. Luminescence responses of recombinant aequorin to 29 metal ions (10^-8-10^-3 M) were characterized by measuring maximum luminescence intensity (I_max), integrated luminescence over 60 s (Int. 60s), and residual luminescence activity following the addition of excess Ca²⁺. Based on luminescence kinetics and recovery of aequorin activity, the responses to metal ions were classified into six categories. Ca²⁺ induced a concentration-dependent flash-type luminescence. Similar flash-type responses were observed with Sr²⁺, Y³⁺, Pb²⁺, and several lanthanide ions, whereas Ba²⁺, Ce³⁺, Ga³⁺, Mg²⁺, Nd³⁺, and V³⁺ induced weak glow-type luminescence without significant loss of aequorin activity. In contrast, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Ni²⁺, and Zn²⁺ induced glow-type luminescence accompanied by reduced residual activity, whereas Cr³⁺ and Zr⁴⁺ produced no luminescence but also decreased aequorin activity. These findings provide a comprehensive quantitative reevaluation of the metal ion selectivity of aequorin and indicate that flash- and glow-type luminescence arise from distinct modes of metal ion interaction with the EF-hand motifs and the resulting conformational changes in aequorin.