Functional Roles of EF-Hands in Human Potassium Channel-Interacting Protein 2.2

Author(s): Liya Lee, Ku-Chung Chen, Long-Sen Chang

Journal Name: Protein & Peptide Letters

Volume 16 , Issue 9 , 2009

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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 Ca2+-binding site of KChIP2.2. However, the Mg2+-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 Ca2+ and Mg2+. 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 Ca2+ and Mg2+.

Keywords: KChIP, Metal-binding properties, EF-hand mutant, Oligomerization

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Article Details

Year: 2009
Page: [1081 - 1087]
Pages: 7
DOI: 10.2174/092986609789055368
Price: $65

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