The lectin-like domain of TNF increases ENaC open probability through a novel site at the interface between the second transmembrane and C-terminal domains of the α-subunit

Rudolf Lucas, Qiang Yue, Abdel Alli, Billie Jeanne Duke, Otor Al-Khalili, Tiffany L. Thai, Jürg Hamacher, Supriya Sridhar, Iryna Lebedyeva, Huabo Su, Susan Tzotzos, Bernhard Fischer, Armanda Formigao Gameiro, Maria Loose, Trinad Chakraborty, Waheed Shabbir, Mohammed Aufy, Rosa Lemmens-Gruber, Douglas C. Eaton, Istvan Czikora

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

Original languageEnglish (US)
Pages (from-to)23440-23451
Number of pages12
JournalJournal of Biological Chemistry
Volume291
Issue number45
DOIs
StatePublished - Nov 4 2016

Fingerprint

Lectins
Peptides
Epithelial Sodium Channels
Membranes
Ubiquitination
Blood pressure
Cell membranes
Ion Channels
Homeostasis
Chemical activation
Binding Sites
Cell Membrane
Blood Pressure
Fluids

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The lectin-like domain of TNF increases ENaC open probability through a novel site at the interface between the second transmembrane and C-terminal domains of the α-subunit. / Lucas, Rudolf; Yue, Qiang; Alli, Abdel; Duke, Billie Jeanne; Al-Khalili, Otor; Thai, Tiffany L.; Hamacher, Jürg; Sridhar, Supriya; Lebedyeva, Iryna; Su, Huabo; Tzotzos, Susan; Fischer, Bernhard; Gameiro, Armanda Formigao; Loose, Maria; Chakraborty, Trinad; Shabbir, Waheed; Aufy, Mohammed; Lemmens-Gruber, Rosa; Eaton, Douglas C.; Czikora, Istvan.

In: Journal of Biological Chemistry, Vol. 291, No. 45, 04.11.2016, p. 23440-23451.

Research output: Contribution to journalArticle

Lucas, R, Yue, Q, Alli, A, Duke, BJ, Al-Khalili, O, Thai, TL, Hamacher, J, Sridhar, S, Lebedyeva, I, Su, H, Tzotzos, S, Fischer, B, Gameiro, AF, Loose, M, Chakraborty, T, Shabbir, W, Aufy, M, Lemmens-Gruber, R, Eaton, DC & Czikora, I 2016, 'The lectin-like domain of TNF increases ENaC open probability through a novel site at the interface between the second transmembrane and C-terminal domains of the α-subunit', Journal of Biological Chemistry, vol. 291, no. 45, pp. 23440-23451. https://doi.org/10.1074/jbc.M116.718163
Lucas, Rudolf ; Yue, Qiang ; Alli, Abdel ; Duke, Billie Jeanne ; Al-Khalili, Otor ; Thai, Tiffany L. ; Hamacher, Jürg ; Sridhar, Supriya ; Lebedyeva, Iryna ; Su, Huabo ; Tzotzos, Susan ; Fischer, Bernhard ; Gameiro, Armanda Formigao ; Loose, Maria ; Chakraborty, Trinad ; Shabbir, Waheed ; Aufy, Mohammed ; Lemmens-Gruber, Rosa ; Eaton, Douglas C. ; Czikora, Istvan. / The lectin-like domain of TNF increases ENaC open probability through a novel site at the interface between the second transmembrane and C-terminal domains of the α-subunit. In: Journal of Biological Chemistry. 2016 ; Vol. 291, No. 45. pp. 23440-23451.
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abstract = "Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.",
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T1 - The lectin-like domain of TNF increases ENaC open probability through a novel site at the interface between the second transmembrane and C-terminal domains of the α-subunit

AU - Lucas, Rudolf

AU - Yue, Qiang

AU - Alli, Abdel

AU - Duke, Billie Jeanne

AU - Al-Khalili, Otor

AU - Thai, Tiffany L.

AU - Hamacher, Jürg

AU - Sridhar, Supriya

AU - Lebedyeva, Iryna

AU - Su, Huabo

AU - Tzotzos, Susan

AU - Fischer, Bernhard

AU - Gameiro, Armanda Formigao

AU - Loose, Maria

AU - Chakraborty, Trinad

AU - Shabbir, Waheed

AU - Aufy, Mohammed

AU - Lemmens-Gruber, Rosa

AU - Eaton, Douglas C.

AU - Czikora, Istvan

PY - 2016/11/4

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N2 - Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val567, Glu568, and Glu571, located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.

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