TNF Lectin-Like Domain Restores Epithelial Sodium Channel Function in Frameshift Mutants Associated with Pseudohypoaldosteronism Type 1B

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.

In silico evidence for prednisone and progesterone efficacy in recurrent implantation failure treatment

“…Progesterone has been shown in some studies to decrease recurrent miscarriage by balancing the Th1/Th2 cytokines [ 24 ]. Several studies have shown that residues Cys101-Glu116 play an essential role at the binding site of TNF-α [ 25 ]. As shown in Fig.…”

CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

“…Additionally, we have also shown that in A549 cells TNF can positively increase single channel kinetics without affecting conductivity of the channel and this increase was completely abolished in PNGase F pretreated cells [ 13 ]. Glycosylation sites on the ENaC subunits are necessary for TNF-induced activation of ENaC current and membrane insertion [ 19 ].…”

Novel and Evolving Therapies for COVID-19 Related Pulmonary Complications,

“…Solnatide mainly activates the pulmonary epithelial sodium channel and restores the injured endothelial-epithelial barrier of pulmonary alveoli. 23,24 In 2013, APEPTICO fruitfully concluded phase 1 clinical study in healthy subjects, verifying the safety of solnatide. Additionally, APEPTICO also effectively finished a randomized, doubleblinded placebo-controlled phase 2 clinical trial in mechanically ventilated ARDS patients with lung oedema, proving the purpose of inhaled solnatide.…”