CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

Waheed Shabbir, Nermina Topcagic, Mohammed Aufy, Murat Oz

    Publications: Contribution to journalArticlePeer Reviewed

    Abstract

    Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

    Original languageEnglish
    Article number1858
    Pages (from-to)1-10
    Number of pages10
    JournalInternational Journal of Molecular Sciences
    Volume22
    Issue number4
    DOIs
    Publication statusPublished - 2 Feb 2021

    Austrian Fields of Science 2012

    • 106002 Biochemistry

    Keywords

    • A549 Cells
    • CRISPR-Cas Systems
    • Epithelial Sodium Channels/genetics
    • Humans
    • Tumor Necrosis Factor-alpha/genetics
    • CRISPR/Cas9
    • Tumor necrosis factor (TNF)
    • Epithelial sodium channel (ENaC)

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