Kv channel interacting proteins (KChIPs) are Ca2+-binding proteins with four EF-hands and well-known to modulate Kv4.2 channel gating. The present study is carried out to investigate the molecular mechanism related to regulate the interaction of KChIP2.2 and KChIP4a with Kv channel. In comparison with KChIP4a, the interaction of KChIP2.2 with Kv4.2 was more obvious in the absence of Ca2+ or Mg2+. However the binding of KChIP2.2 and KChIP4a toward Kv4.2 increased with increasing Ca2+ and Mg2+ concentration. Nevertheless, no individual regions within KChIP2.2 and KChIP4a could exclusively fulfill the interaction between KChIPs mutants and Kv channel. Fluorescence measurement showed that KChIP2.2 possessed both high affinity and low affinity Ca2+-binding sites, but only low affinity Ca2+-binding site was observed with KChIP4a. However, both of them have only one Mg2+-binding site. Studies on the truncated mutants revealed that the EF-hand 4 of KChIP2.2 was related to high affinity binding with Ca2+, and the integrity of molecular structure of KChIP2.2 and KChIP4a was important for Ca2+ -and Mg2+-binding. The thermal stability of KChIP2.2 and KChIP4 was found to be differentiately affected by Ca2+ and Mg2+. Proteolytic digestion and thiol reactivity assays also supported that Ca2+ and Mg2+-induced conformational change of KChIP2.2 was differed from KChIP4a. Moreover, in cells co-transfected with Kv4.2 cDNA, it was formed that KChIP2.2 trafficking to the cell surface was increased by elevating intracellular Ca2+ concentration, but no noticeable change was observed for KChIP4a. Taken together, these results suggest that the conformational changes of KChIP2.2 and KChIP4a differently induced by Ca2+ and Mg2+ affect their binding with Kv channel and/or cellular distribution.

Mutagenesis studies on the four EF-hands of KChIP2.2 (Potassium channel-interacting protein 2.2) were carried out to explore the conformational transition upon the binding of Ca2+ and Mg2+ and the subsequent effect on the interaction between KChIP2.2 and Kv4.2. CD spectra indicated that Ca2+- and Mg2+-loaded wild-type and mutated KChIP2.2 altered the secondary structure contents. In contrast to other mutants, mutation on EF1 caused a notably change in the secondary structure of KChIP2.2. Fluorescence measurement revealed that EF-hands 3 and 4 were high affinity Ca2+-binding sites within KChIP2.2 molecule, but the binding of Mg2+ with KChIP2.2 was marginally affected by EF-hand mutations. The results of size-exclusion chromatography showed that mutations on EF-1, EF-2 and EF-3 induced the oligomerization of KChIP2.2 and the extent of oligomerization was enhanced by Ca2+ and Mg2+. No significant differences were noted when wild-type and mutated KChIP2.2 bound with porcine brain membrane and liposome either in the absence or presence of Ca2+- and Mg2+. Pull down assay showed that KChIP2.2 and EF-hand mutants could bind with Kv4.2 in the absence of Ca2+ and Mg2+, but the interaction was enhanced by Ca2+ and Mg2+. However, the binding capability of mutants for Kv4.2 was notably lower than that observed for wild-type KChIP2.2. It was found that, in sharp contrast to that EF1 mutant exclusively localized in the nucleus, the other EF-hand mutants and wild-type protein distributed within nucleus as well as cytoplasm. Elevating intracellular Ca2+ concentration caused the translocation of EF1 mutant to cytoplasm but no appreciable effect on other mutants and wild-type KChIP2.2. . Taken together, these results suggest that the integrity of the four EF-hands are involved in function to stabilize conformation for binding with Kv channel, but this conformational transition is not essential for the binding to cell membrane.