Restoration of Epithelial Sodium Channel Function by Synthetic Peptides in Pseudohypoaldosteronism Type 1B Mutants.

Anita Willam,Susan Tzotzos,Sabine Chytracek,Waheed Shabbir,Heinrich Evanzin,Helmut Pietschmann,Rosa Lemmens-Gruber,Hendrik Fischer,Sabrina Geppert,Mohammed Aufy,Bernhard Fischer,Rudolf Lucas,Istvan Czikora

doi:10.3389/fphar.2017.00085

Anita Willam, Susan Tzotzos + Show 11 more

Open Access

https://doi.org/10.3389/fphar.2017.00085

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Abstract

The synthetically produced cyclic peptides solnatide (a.k.a. TIP or AP301) and its congener AP318, whose molecular structures mimic the lectin-like domain of human tumor necrosis factor (TNF), have been shown to activate the epithelial sodium channel (ENaC) in various cell- and animal-based studies. Loss-of-ENaC-function leads to a rare, life-threatening, salt-wasting syndrome, pseudohypoaldosteronism type 1B (PHA1B), which presents with failure to thrive, dehydration, low blood pressure, anorexia and vomiting; hyperkalemia, hyponatremia and metabolic acidosis suggest hypoaldosteronism, but plasma aldosterone and renin activity are high. The aim of the present study was to investigate whether the ENaC-activating effect of solnatide and AP318 could rescue loss-of-function phenotype of ENaC carrying mutations at conserved amino acid positions observed to cause PHA1B. The macroscopic Na+ current of all investigated mutants was decreased compared to wild type ENaC when measured in whole-cell patch clamp experiments, and a great variation in the membrane abundance of different mutant ENaCs was observed with Western blotting experiments. However, whatever mechanism leads to loss-of-function of the studied ENaC mutations, the synthetic peptides solnatide and AP318 could restore ENaC function up to or even higher than current levels of wild type ENaC. As therapy of PHA1B is only symptomatic so far, the peptides solnatide and AP318, which directly target ENaC, are promising candidates for the treatment of the channelopathy-caused disease PHA1B.

Highlights

The amiloride-sensitive epithelial sodium channel (ENaC), a member of the ENaC/degenerin (ENaC/DEG) family of ion channels (Canessa et al, 1993; Lingueglia et al, 1993; Kellenberger and Schild, 2015), is responsible for the maintenance of Na+ balance, extracellular fluid volume and blood pressure (Garty and Palmer, 1997)
The only difference between solnatide and AP318 is that the disulphide bridge between cysteines in positions 1 and 17 and which links the N- and C-termini in solnatide has been replaced by an amide bond linking the amino group of 4-amino-butanoic acid in position 1 with the side chain carboxyl group of aspartic acid in position 17 (Figure 1)
The γV543fs mutation is predicted to result in synthesis of a truncated translation product of 597 amino acid residues with an abnormal sequence after S542

Summary

Introduction

The amiloride-sensitive epithelial sodium channel (ENaC), a member of the ENaC/degenerin (ENaC/DEG) family of ion channels (Canessa et al, 1993; Lingueglia et al, 1993; Kellenberger and Schild, 2015), is responsible for the maintenance of Na+ balance, extracellular fluid volume and blood pressure (Garty and Palmer, 1997). ENaC is located at the apical membrane of salt-reabsorbing tight epithelia of the distal nephron, the distal colon, salivary and sweat glands and the lung, where it constitutes the rate-limiting step for vectorial movement of Na+ ions from the luminal side into the cell interior; the basolaterally located Na,K-ATPase actively transports Na+ out of the cell, providing the driving force for Na+ reabsorption (Kellenberger and Schild, 2002). In the kidney and the colon, Na+ reabsorption through ENaC, stimulated by the mineralocorticoid hormone aldosterone, is crucial for the maintenance of blood Na+ and K+ levels and their homeostasis (Kellenberger and Schild, 2002). ENaC-mediated Na+ absorption from the intestinal lumen is under glucocorticoid control (Schild, 2010)

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