The Epithelial Sodium Channel-An Underestimated Drug Target

Abstract

Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents.

Keywords: Liddle syndrome; TIP peptides; cystic fibrosis; epithelial sodium channel; gain- and loss-of-function mutations; pseudohypoaldosteronism type 1B; setanaxib; solnatide.

Figure 1

ENaC activation by intra- and extracellular proteases. (A) Cartoon illustration of ENaC cleavage by furin within the trans-Golgi network and extracellular proteases at the cell surface. P_O, open probability. (B) Peptide sequences of the α- and γ-subunits of human ENaC showing the regions containing the inhibitory peptides within the extracellular loop. Furin consensus sites are shown in blue; the inhibitory peptides [38] are shown in red. The figure shows extracellular proteases cleaving the γ-subunit whose cleavage sites have been identified by mutagenesis studies [48,72,73]: Serine proteases are shown in magenta, cysteine protease in orange, and metalloprotease in brown. CAP, channel-activating protease (alternative name for the indicated proteases); FSAP-SPD, factor VII activating protease—serine protease domain. Reproduced from [74] (Figure 1); Copyright © The Authors, 2021; published by Springer, Berlin, New York.

Figure 2

ENaC subunit cysteines (mouse ENaC). (A) A Schematic of an ENaC subunit, adapted from [5]. Cysteine residues are represented by yellow circles, and disulfide bonds observed in the ASIC1 structure are represented by black bars. Conserved aspartic acids αAsp-171 and γAsp-115 are represented by a red circle, and αTyr-474 is represented by a blue circle. (B) B Sequence alignment of finger domain segment hosting conserved cysteines in ENaC subunits. (C) C Sequence alignment of the thumb domain residues proximal to the finger domain in the ASIC1 structure. Identities are shaded gray. Cysteines are shaded black. Reproduced from [83] (Figure 1); Copyright © 2023 by The American Society for Biochemistry and Molecular Biology, Inc.; published by American Society for Biochemistry and Molecular Biology, Baltimore, MD, USA.

Figure 3

Potential sites of phosphorylation in α-ENaC C-terminal region. Two serine residues and one proline residue are highly conserved in a C-terminal region of α-ENaC close to the second transmembrane domain. (A) Schematic representation of α-ENaC illustrating the extracellular loop, two transmembrane domains (M1 and M2) and intracellular N- and C-termini. The amino acid sequence of rat α-ENaC (residues 613–625) corresponds to the C-terminal region indicated by a star (*) and contains the serine residues 617 (S617) and 621 (S621) and the proline residue 622 (P622) highlighted in bold. (B) Amino acid sequence alignment of this highly conserved C-terminal region from several mammalian α-ENaC subunits. The residues homologous to S617, S621 and P622 in rat αENaC are highlighted in bold. (C) Amino acid sequence alignment of homologous C-terminal regions from human β-, γ- and δ-ENaC subunits, showing a lack of potential phosphorylation sites. Adapted from [110] (Figure 1); Copyright © The Author(s) 2022, corrected publication 2022; published by Springer Nature.

Figure 4

PHA-1B patient with miliaria rubra on the face. Reproduced from [145] (Figure 1); Copyright © The Author(s) 2021; published by BioMed Central, London.

Figure 5

Simplified scheme of ENaC regulation and targets for ENaC modulation. Abbreviations: ACE-I (angiotensin-converting enzyme inhibitors), Aldo (aldosterone), AngII (angiotensin II), Cx30 (connexin 30), MLP-1 (myristoylated alanine-rich C kinase (MARCKS)-like protein 1), MR (mineralcorticoid receptor), N (asparagine), Nedd 4-2 (neural precursor cell expressed developmentally down-regulated 4-2), NOXA1 (reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activator 1), PIP2 (phosphatidylinositol 4,5-bisphosphate), PIP3 (phosphatidylinositol 4,5-triphosphate), PKC-α (proteinkinase C isoform α), siRNA (short interfering ribonucleic acid), SPLUNC1 (short palate, lung, and nasal epithelial clone 1), ROS (reactive oxygene species), SGK1 (serum and glucocorticoid-regulated kinase 1), ub (ubiquitination). Red triangles indicate phosphorylation.