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 α-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 α-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.
Highlights
Tumor necrosis factor (TNF) is a mammalian inflammatory cytokine, which exerts a plethora of effects primarily aimed at defending the host against invading pathogens
In Czikora’s study, using a cyclic peptide which mimics the lectin-like domain of TNF, it was not demonstrated directly that native TNF can activate ENaC via these proposed mechanisms
We have previously shown that the synthetic cyclic peptide solnatide, which mimics the lectin-like domain of TNF, requires one of the two pore-forming α- and δ-ENaC subunits to induce its maximum amiloride-sensitive sodium current-activating effect [16]
Summary
Tumor necrosis factor (TNF) is a mammalian inflammatory cytokine, which exerts a plethora of effects primarily aimed at defending the host against invading pathogens. TNF is crucially involved in the control of Trypanosoma brucei brucei and T. cruzei infections, through the trypanolytic effect triggered by interaction of its lectin-like domain with the N-linked N,N′-diacetylchitobiose core of the variant surface glycoproteins (VSG) of these organisms [1, 2, 6,7,8,9,10,11,12,13,14]. Transgenic mice expressing a mutated TNF lectin-like domain are more prone to develop lung edema than their wild-type (WT) counterparts when challenged with the bacterial toxin pneumolysin (PLY) [19]
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