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

Waheed Shabbir,Nermina Topcagic,Murat Oz,Mohammed Aufy

doi:10.3390/ijms22041858

Waheed Shabbir, Nermina Topcagic + Show 2 more

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https://doi.org/10.3390/ijms22041858

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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.

Highlights

To analyze the role of δ-epithelial Na+ channel (ENaC) in tumor necrosis factor (TNF)-mediated activation of ENaC, we knocked down the δ-ENaC with CRISPR/Cas 9 plasmids in A549 cells
The δ-ENaC CRISPR/Cas 9 KO plasmids decreased 90% of the δ-ENaC protein expression in A549 cell compared with control CRISPR/Cas 9 plasmid, as shown in western blot and immunofluorescence assays (Figure 1A,B)
These results indicate that δ-ENaC CRISPR/Cas 9 KO plasmid significantly knocked down the δ-ENaC

Summary

Introduction

Four epithelial sodium channel (ENaC α, β, γ and δ subunits) have been cloned in mammals [1,2,3]. Δ-ENaC expression has been found in sevral human tissues including gonads, pancreas, brain, heart, liver and thymus, with lower amounts in kidney and lung [3,4]. The function of δ-ENaC subunits in these tissues are not well studied. Some studies have shown that δENaC containing ENaC have very slow activation and desensitization kinetics in response to decrease in extracellular pH [5] supporting their role as slow pH sensors which may be found in ischemia [4]. Δ-ENaC channels was shown to contribute ~50% of amiloridesensitive salt transport across primary human nasal epithelial cells [6] Some studies have shown that δENaC containing ENaC have very slow activation and desensitization kinetics in response to decrease in extracellular pH [5] supporting their role as slow pH sensors which may be found in ischemia [4]. δ-ENaC channels was shown to contribute ~50% of amiloridesensitive salt transport across primary human nasal epithelial cells [6]

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