Cadmium Trapping in an Epithelial Sodium Channel Pore Mutant

Abstract

The putative selectivity filter of the epithelial sodium channel (ENaC) comprises a three-residue sequence G/SXS, but it remains uncertain whether the backbone atoms of this sequence or whether their side chains are lining the pore. It has been reported that the S589C mutation in the selectivity filter of ␣ENaC renders the channel sensitive to block by externally applied Cd 2؉ ; this was interpreted as evidence for Cd 2؉ coordination with the thiol group of the side chain of ␣589C, pointing toward the pore lumen. Because the ␣S589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd 2؉ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of ␣S589C ENaC by Cd 2؉ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd 2؉ inhibits not only ENaC ␣S589C and ␣S589D but also ␣S589N mutants and that Ca 2؉ weakly interacts with the S589D mutant. The block of ␣S589C, -D, and -N mutants is characterized by a slow on-rate, is nearly irreversible, is voltage-dependent, and can be prevented by amiloride. The C546S mutation in the second transmembrane helix of ␥ subunit in the background of the ENaC ␣S589C, -D, or -N mutants reduces the sensitivity to block by Cd 2؉ and renders the block rapidly reversible. We conclude therefore that the block by Cd 2؉ of the ␣S589C, -D, and -N mutants results from the trapping of Cd 2؉ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the ␥ENaC subunit.The epithelial sodium channel (ENaC) 3 mediates the Na ϩ ion influx across the apical membrane of tight epithelia such as the aldosterone-sensitive distal nephron, the distal colon, or the airways epithelium (1). In principal cells of the aldosteronesensitive distal nephron, the ENaC-mediated influx of Na ϩ ions constitutes the apical step of the transepithelial Na ϩ absorption and is regulated by aldosterone (2). The aldosterone-sensitive distal nephron plays an important role in the maintenance of Na ϩ homeostasis, the regulation of extracellular volume, and blood pressure (3). In the lungs, ENaC together with cystic fibrosis transmembrane conductance regulator controls the height of the airway surface liquid that forms a thin layer coating the airways epithelium, which contributes to the clearance of particles and microbes (4).Functional ENaC at the cell plasma membrane is a heteromeric protein made of homologous ␣, ␤, and ␥ subunits arranged pseudosymetrically around a central channel pore, presumably in a 2 ␣, 1 ␤, and 1 ␥ configuration (5, 6). Each ENaC subunit is made of two transmembrane helices (TM1 and TM2), a large extracellular loop that represents more than half of the mass of the protein, and intracellular N and C termini. The ENaC subunits belong to the ENaC/degenerin superfamily of ion channels but share no sequence homology with other tetrameric channels also made of two transmembrane domains, such as inward rect...

Pseudohypoaldosteronism type 1 and Liddle's syndrome mutations that affect the single-channel properties of the epithelial Na+channel

“…,b; Takeda et al. ). As shown in the model presented in Figure of an archetype ENaC subunit, which is based upon the chicken ASIC1 crystal structure as solved by the Gouaux laboratory (Jasti et al.…”

“…,b; Takeda et al. ). Homologous series (S445 in cASIC1) at this position are highly conserved across all Deg/ENaC subunits and are recognized to contribute critical structure to the selectivity filter of channels formed by these proteins.…”

“…In this latter case, mutation of S562 likely results in a nonfunctional channel with a disorganized conduction pathway. The TM2s of the three symmetry-related subunits that comprise heterotrimeric ENaC, a, b, and c, form the conduction pore of the channel through the plasma membrane (Sheng et al 2001a,b;Takeda et al 2007). As shown in the model presented in Figure 5 of an archetype ENaC subunit, which is based upon the chicken ASIC1 crystal structure as solved by the Gouaux laboratory (Jasti et al 2007;Gonzales et al 2009;Baconguis and Gouaux 2012;Baconguis et al 2013Baconguis et al , 2014, N530 is located in TM2 of the cENaC subunit near the extracellular mouth of the pore.…”

International Union of Basic and Clinical Pharmacology. XCI. Structure, Function, and Pharmacology of Acid-Sensing Ion Channels and the Epithelial Na+Channel

“…Substitution of the C-terminal Ser residue of the conserved G/S-x-S motif in the lower part of the TM2 (Ser589 in a ENaC) with large residues reduced the high selectivity of ENaC for Na + ions considerably (Kellenberger et al, 1999a). The likely explanation for this observation is that the mutations of a ENaC-S589 22 Kellenberger and Schild enlarge the pore diameter, allowing large cations, such as K + or ammonium, to permeate the ENaC pore (Kellenberger et al, 2001;Takeda et al, 2007). These and related studies identified the ion selectivity filter as the narrowest part of the ENaC channel pore, allowing almost exclusively monovalent cations of the size of Na + to pass through the channel pore (Kellenberger et al, 1999b;Snyder et al, 1999;Sheng et al, 2000).…”

Mast Cell Chemotaxis – Chemoattractants and Signaling Pathways

“…Mammalian target of rapamycin complexes (mTORCs) were found to play important roles in chemotaxis of several cell models such as neutrophils (Charest et al, 2010; Liu et al, 2010) and Dictyostelium (Sasaki and Firtel, 2006; Takeda et al, 2007; Liu and Parent, 2011). mTORC1 is activated in PI3K-dependent manner and its inhibition by rapamycin depressed the SCF-mediated migration (Kim et al, 2008a,b).…”