Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome

Qiuming Gong, Melanie A. Jones, Zhengfeng Zhou

    Research output: Contribution to journalArticle

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    Abstract

    Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.

    Original languageEnglish (US)
    Pages (from-to)4069-4074
    Number of pages6
    JournalJournal of Biological Chemistry
    Volume281
    Issue number7
    DOIs
    StatePublished - Feb 17 2006

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    Human Trafficking
    Long QT Syndrome
    Calnexin
    Ether
    Genes
    Pharmacology
    Endoplasmic Reticulum
    Association reactions
    Trypsin
    Digestion
    Glycosylation
    Brefeldin A
    Mutation
    Cell membranes
    Cell Membrane
    E 4031

    ASJC Scopus subject areas

    • Biochemistry

    Cite this

    Mechanisms of pharmacological rescue of trafficking-defective hERG mutant channels in human long QT syndrome. / Gong, Qiuming; Jones, Melanie A.; Zhou, Zhengfeng.

    In: Journal of Biological Chemistry, Vol. 281, No. 7, 17.02.2006, p. 4069-4074.

    Research output: Contribution to journalArticle

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    abstract = "Long QT syndrome type 2 is caused by mutations in the human ether-a-go-go-related gene (hERG). We previously reported that the N470D mutation is retained in the endoplasmic reticulum (ER) but can be rescued to the plasma membrane by hERG channel blocker E-4031. The mechanisms of ER retention and how E-4031 rescues the N470D mutant are poorly understood. In this study, we investigated the interaction of hERG channels with the ER chaperone protein calnexin. Using coimmunoprecipitation, we showed that the immature forms of both wild type hERG and N470D associated with calnexin. The association required N-linked glycosylation of hERG channels. Pulse-chase analysis revealed that N470D had a prolonged association with calnexin compared with wild type hERG and E-4031 shortened the time course of calnexin association with N470D. To test whether the prolonged association of N470D with calnexin is due to defective folding of mutant channels, we studied hERG channel folding using the trypsin digestion method. We found that N470D and the immature form of wild type hERG were more sensitive to trypsin digestion than the mature form of wild type hERG. In the presence of E-4031, N470D became more resistant to trypsin even when its ER-to-Golgi transport was blocked by brefeldin A. These results suggest that defective folding of N470D contributes to its prolonged association with calnexin and ER retention and that E-4031 may restore proper folding of the N470D channel leading to its cell surface expression.",
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