To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg2+, Ca2+, Sr2+, and Ba2+) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca2+-binding sites, high-affinity and low-affinity Ca2+-binding sites. However, only low-affinity-binding site for Mg2+, Sr2+, and Ba2+ was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca2+-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1. (C) 2003 Elsevier Inc. All rights reserved.