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Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis

Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC functi...

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Autores principales: Ilyaskin, Alexandr V., Korbmacher, Christoph, Diakov, Alexei
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Biochemistry and Molecular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973139/
https://www.ncbi.nlm.nih.gov/pubmed/33577799
http://dx.doi.org/10.1016/j.jbc.2021.100404
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author Ilyaskin, Alexandr V.
Korbmacher, Christoph
Diakov, Alexei
author_facet Ilyaskin, Alexandr V.
Korbmacher, Christoph
Diakov, Alexei
author_sort Ilyaskin, Alexandr V.
collection PubMed
description Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency.
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spelling pubmed-79731392021-03-19 Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis Ilyaskin, Alexandr V. Korbmacher, Christoph Diakov, Alexei J Biol Chem Research Article Mice lacking connexin 30 (Cx30) display increased epithelial sodium channel (ENaC) activity in the distal nephron and develop salt-sensitive hypertension. This indicates a functional link between Cx30 and ENaC, which remains incompletely understood. Here, we explore the effect of Cx30 on ENaC function using the Xenopus laevis oocyte expression system. Coexpression of human Cx30 with human αβγENaC significantly reduced ENaC-mediated whole-cell currents. The size of the inhibitory effect on ENaC depended on the expression level of Cx30 and required Cx30 ion channel activity. ENaC inhibition by Cx30 was mainly due to reduced cell surface ENaC expression resulting from enhanced ENaC retrieval without discernible effects on proteolytic channel activation and single-channel properties. ENaC retrieval from the cell surface involves the interaction of the ubiquitin ligase Nedd4-2 with PPPxY-motifs in the C-termini of ENaC. Truncating the C- termini of β- or γENaC significantly reduced the inhibitory effect of Cx30 on ENaC. In contrast, mutating the prolines belonging to the PPPxY-motif in γENaC or coexpressing a dominant-negative Xenopus Nedd4 (xNedd4-CS) did not significantly alter ENaC inhibition by Cx30. Importantly, the inhibitory effect of Cx30 on ENaC was significantly reduced by Pitstop-2, an inhibitor of clathrin-mediated endocytosis, or by mutating putative clathrin adaptor protein 2 (AP-2) recognition motifs (YxxФ) in the C termini of β- or γ-ENaC. In conclusion, our findings suggest that Cx30 inhibits ENaC by promoting channel retrieval from the plasma membrane via clathrin-dependent endocytosis. Lack of this inhibition may contribute to increased ENaC activity and salt-sensitive hypertension in mice with Cx30 deficiency. American Society for Biochemistry and Molecular Biology 2021-02-10 /pmc/articles/PMC7973139/ /pubmed/33577799 http://dx.doi.org/10.1016/j.jbc.2021.100404 Text en © 2021 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Research Article
Ilyaskin, Alexandr V.
Korbmacher, Christoph
Diakov, Alexei
Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title_full Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title_fullStr Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title_full_unstemmed Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title_short Inhibition of the epithelial sodium channel (ENaC) by connexin 30 involves stimulation of clathrin-mediated endocytosis
title_sort inhibition of the epithelial sodium channel (enac) by connexin 30 involves stimulation of clathrin-mediated endocytosis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7973139/
https://www.ncbi.nlm.nih.gov/pubmed/33577799
http://dx.doi.org/10.1016/j.jbc.2021.100404
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