TY - JOUR
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
N1 - Funding Information:
This work was funded by Wellcome Trust Pathfinder Award 105632 and Wirtschaftsagentur Wien Call FemPower 2015 ID 1458688. AW and WS also received financial support from APEPTICO RandD Vienna, Austria. RL was supported by an NIH RO1 DK100564 RO1 grant and IC received an AHA postdoctoral award 15POST22820021.
Publisher Copyright:
© 2017 Willam, Aufy, Tzotzos, El-Malazi, Poser, Wagner, Unterköfler, Gurmani, Martan, Iqbal, Fischer, Fischer, Pietschmann, Czikora, Lucas, Lemmens-Gruber and Shabbir.
PY - 2017/5/29
Y1 - 2017/5/29
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.
AB - 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.
KW - Amiloride-sensitive epithelial sodium channel
KW - Lectin-like domain of tumor necrosis factor
KW - Pseudohypoaldosteronism type 1B
KW - Solnatide (AP301)
KW - TIP peptides
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U2 - 10.3389/fimmu.2017.00601
DO - 10.3389/fimmu.2017.00601
M3 - Article
AN - SCOPUS:85020011180
SN - 1664-3224
VL - 8
JO - Frontiers in immunology
JF - Frontiers in immunology
IS - MAY
M1 - 601
ER -