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Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1

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Abstract

The aim of the present study is to explore whether membrane targeting of K+ channel-interacting protein 1 (KChIP1) is associated with its EF-hand motifs and varies with specific phospholipids. Truncated KChIP1, in which the EFhands 3 and 4 were deleted, retained the α-helix structure, indicating that the N-terminal half of KChIP1 could fold appropriately. Compared with wild-type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked loss of truncated KChIP1, suggesting that intact EF-hands 3 and 4 were crucial for the anchorage of KChIP1 on membrane. KChIP1 showed a higher binding capability with phosphatidylserine (PS) than truncated KChIP1. Unlike that of truncated KChIP1, the binding of wild-type KChIP1 with membrane was enhanced by increasing the PS content. Moreover, the binding of KChIP1 with phospholipid vesicles induced a change in the structure of KChIP1 in the presence of PS. Taken together, our data suggest that EF-hands 3 and 4 of KChIP1 are functionally involved in a specific association with PS on the membrane.

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Abbreviations

ANS:

8-Anilino-1-naphthalene sulphonic acid

BSA:

bovine serum albumin

CD:

circular dichroism

DMEM:

Dulbecco modified Eagle medium

ELISA:

enzyme-linked immunosorbent assay

FTIR:

Fourier transform infrared

GCAP:

guanylyl cyclase-activating protein

GFP:

green fluorescent protein

HRP:

horseradish peroxidase

KChIP:

K+ channel-interacting protein

MLV:

multilamellar large vesicles

NCS:

neuronal calcium sensor

ORF:

open reading frame

PA:

phosphatidic acid

PC:

phosphatidylcholine

PCR:

polymerase chain reaction

PE:

phosphatidylethanolamine

PI:

phosphatidylinositol

PMSF:

phenylmethylsulphonyl fluoride

PS:

phosphatidylserine

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Authors and Affiliations

  1. Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung, 804, Taiwan

    Yan-Shun Liao, Ku-Chung Chen & Long-Sen Chang

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  1. Yan-Shun Liao
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  2. Ku-Chung Chen
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  3. Long-Sen Chang
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Correspondence to Long-Sen Chang.

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Liao, YS., Chen, KC. & Chang, LS. Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1. J Biosci 34, 203–211 (2009). https://doi.org/10.1007/s12038-009-0024-4

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  • DOI: https://doi.org/10.1007/s12038-009-0024-4

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