title: Functional roles of EF-hands in human potassium channel-interacting protein 2.2.
abstract: Single site-directed mutations at each of the four EF-hand loops of potassium channel-interacting protein 2.2 (KChIP2.2) were carried out to explore the functional roles of EF-hands in KChIP2.2. In contrast to those on EF-hands 1 and 2, mutations on EF-hands 3 or 4 distorted the high affinity Ca(2+)-binding site of KChIP2.2. However, the Mg(2+)-binding ability of KChIP2.2 was marginally affected by the mutations. The gross conformation of mutated KChIP2.2 was indistinguishable from wild-type KChIP2.2 as revealed by CD spectra. The results of size exclusion chromatography showed that, with exception of EF-hand 4 mutant, mutations on EF-hands 1, 2 or 3 caused KChIP2.2 to form oligomer. Pull-down assay revealed that, unlike wild-type KChIP2.2, the interaction between mutated KChIP2.2 and Kv4.2 was not notably enhanced by Ca(2+) and Mg(2+). Coexpression of Kv4.2 and KChIP2.2 in HeLa cells revealed that mutations on EF-hands did not alter the intracellular co-localization of KChIP2.2 and Kv4.2. Together with previous findings that EF-hand mutants of KChIP proteins are unable to regulate the kinetics of Kv4.2, our data show that the intact EF-hands should be crucial for the formation of active conformation of KChIP2.2 when the protein is loaded with Ca(2+) and Mg(2+).
authors.affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.
authors.forename: Liya
authors.initials: L
authors.lastname: Lee
authors.forename: Ku-Chung
authors.initials: KC
authors.lastname: Chen
authors.forename: Long-Sen
authors.initials: LS
authors.lastname: Chang
mesh_terms: D002118:Calcium / Q000378:metabolism; D002942:Circular Dichroism; D020832:EF Hand Motifs / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051605:Kv Channel-Interacting Proteins / Q000235:genetics / Q000502:physiology*; D008274:Magnesium / Q000378:metabolism; D051672:Shal Potassium Channels / Q000235:genetics; D013050:Spectrometry, Fluorescence
chemical_list: C496752:KCNIP2 protein, human; D051605:Kv Channel-Interacting Proteins; D051672:Shal Potassium Channels; D008274:Magnesium; D002118:Calcium