TNF lectin-like domain restores epithelial sodium channel function in frameshift mutants associated with pseudohypoaldosteronism type 1B

Anita Willam, Mohammed Aufy, Susan Tzotzos, Dina El-Malazi, Franziska Poser, Alina Wagner, Birgit Unterköfler, Didja Gurmani, David Martan, Shahid Muhammad Iqbal, Bernhard Fischer, Hendrik Fischer, Helmut Pietschmann, Istvan Czikora, Rudolf Lucas, Rosa Lemmens-Gruber, Waheed Shabbir

Research output: Contribution to journalArticle

Abstract

Previous in vitro studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the a-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane abundance varied between mutants. Solnatide restored function in α-ENaC frameshift mutants to current density levels of wild type ENaC or higher despite their lacking a binding site for solnatide, previously located to the region between TM2 and the C-terminus of the a-subunit. TNF similarly restored current density to wild type levels in the mutant αR448fs. Activation of βγ-ENaC may contribute to this moderate current enhancement, but whatever the mechanism, experimental data indicate that solnatide could be a new strategy to treat PHA1B.

Original languageEnglish (US)
Article number601
JournalFrontiers in immunology
Volume8
Issue numberMAY
DOIs
StatePublished - May 29 2017

Fingerprint

Pseudohypoaldosteronism
Epithelial Sodium Channels
Lectins
Tumor Necrosis Factor-alpha
Glycosylation
Wasting Syndrome
Cyclic Peptides
Phase II Clinical Trials
Frameshift Mutation
Membranes
Amiloride
Molecular Structure

Keywords

  • Amiloride-sensitive epithelial sodium channel
  • Lectin-like domain of tumor necrosis factor
  • Pseudohypoaldosteronism type 1B
  • Solnatide (AP301)
  • TIP peptides

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

Cite this

TNF lectin-like domain restores epithelial sodium channel function in frameshift mutants associated with pseudohypoaldosteronism type 1B. / Willam, Anita; Aufy, Mohammed; Tzotzos, Susan; El-Malazi, Dina; Poser, Franziska; Wagner, Alina; Unterköfler, Birgit; Gurmani, Didja; Martan, David; Iqbal, Shahid Muhammad; Fischer, Bernhard; Fischer, Hendrik; Pietschmann, Helmut; Czikora, Istvan; Lucas, Rudolf; Lemmens-Gruber, Rosa; Shabbir, Waheed.

In: Frontiers in immunology, Vol. 8, No. MAY, 601, 29.05.2017.

Research output: Contribution to journalArticle

Willam, A, Aufy, M, Tzotzos, S, El-Malazi, D, Poser, F, Wagner, A, Unterköfler, B, Gurmani, D, Martan, D, Iqbal, SM, Fischer, B, Fischer, H, Pietschmann, H, Czikora, I, Lucas, R, Lemmens-Gruber, R & Shabbir, W 2017, 'TNF lectin-like domain restores epithelial sodium channel function in frameshift mutants associated with pseudohypoaldosteronism type 1B', Frontiers in immunology, vol. 8, no. MAY, 601. https://doi.org/10.3389/fimmu.2017.00601
Willam, Anita ; Aufy, Mohammed ; Tzotzos, Susan ; El-Malazi, Dina ; Poser, Franziska ; Wagner, Alina ; Unterköfler, Birgit ; Gurmani, Didja ; Martan, David ; Iqbal, Shahid Muhammad ; Fischer, Bernhard ; Fischer, Hendrik ; Pietschmann, Helmut ; Czikora, Istvan ; Lucas, Rudolf ; Lemmens-Gruber, Rosa ; Shabbir, Waheed. / TNF lectin-like domain restores epithelial sodium channel function in frameshift mutants associated with pseudohypoaldosteronism type 1B. In: Frontiers in immunology. 2017 ; Vol. 8, No. MAY.
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T1 - TNF lectin-like domain restores epithelial sodium channel function in frameshift mutants associated with pseudohypoaldosteronism type 1B

AU - Willam, Anita

AU - Aufy, Mohammed

AU - Tzotzos, Susan

AU - El-Malazi, Dina

AU - Poser, Franziska

AU - Wagner, Alina

AU - Unterköfler, Birgit

AU - Gurmani, Didja

AU - Martan, David

AU - Iqbal, Shahid Muhammad

AU - Fischer, Bernhard

AU - Fischer, Hendrik

AU - Pietschmann, Helmut

AU - Czikora, Istvan

AU - Lucas, Rudolf

AU - Lemmens-Gruber, Rosa

AU - Shabbir, Waheed

PY - 2017/5/29

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N2 - Previous in vitro studies have indicated that tumor necrosis factor (TNF) activates amiloride-sensitive epithelial sodium channel (ENaC) current through its lectin-like (TIP) domain, since cyclic peptides mimicking the TIP domain (e.g., solnatide), showed ENaC-activating properties. In the current study, the effects of TNF and solnatide on individual ENaC subunits or ENaC carrying mutated glycosylation sites in the a-ENaC subunit were compared, revealing a similar mode of action for TNF and solnatide and corroborating the previous assumption that the lectin-like domain of TNF is the relevant molecular structure for ENaC activation. Accordingly, TNF enhanced ENaC current by increasing open probability of the glycosylated channel, position N511 in the α-ENaC subunit being identified as the most important glycosylation site. TNF significantly increased Na+ current through ENaC comprising only the pore forming subunits α or δ, was less active in ENaC comprising only β-subunits, and showed no effect on ENaC comprising γ-subunits. TNF did not increase the membrane abundance of ENaC subunits to the extent observed with solnatide. Since the α-subunit is believed to play a prominent role in the ENaC current activating effect of TNF and TIP, we investigated whether TNF and solnatide can enhance αβγ-ENaC current in α-ENaC loss-of-function frameshift mutants. The efficacy of solnatide has been already proven in pathological conditions involving ENaC in phase II clinical trials. The frameshift mutations αI68fs, αT169fs, αP197fs, αE272fs, αF435fs, αR438fs, αY447fs, αR448fs, αS452fs, and αT482fs have been reported to cause pseudohypoaldosteronism type 1B (PHA1B), a rare, life-threatening, salt-wasting disease, which hitherto has been treated only symptomatically. In a heterologous expression system, all frameshift mutants showed significantly reduced amiloride-sensitive whole-cell current compared to wild type αβγ-ENaC, whereas membrane abundance varied between mutants. Solnatide restored function in α-ENaC frameshift mutants to current density levels of wild type ENaC or higher despite their lacking a binding site for solnatide, previously located to the region between TM2 and the C-terminus of the a-subunit. TNF similarly restored current density to wild type levels in the mutant αR448fs. Activation of βγ-ENaC may contribute to this moderate current enhancement, but whatever the mechanism, experimental data indicate that solnatide could be a new strategy to treat PHA1B.

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KW - TIP peptides

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