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 alphaENaC renders the channel sensitive to block by externally applied Cd2+; this was interpreted as evidence for Cd2+ coordination with the thiol group of the side chain of alpha589C, pointing toward the pore lumen. Because the alphaS589C mutation alters the monovalent to divalent cation selectivity ratio of ENaC and because internally applied Cd2+ blocks wild-type ENaC with high affinity, we hypothesized that the inhibition of alphaS589C ENaC by Cd2+ results rather from the coordination of this cation with native cysteine residues located in the internal pore of ENaC. We show here that Cd2+ inhibits not only ENaC alphaS589C and alphaS589D but also alphaS589N mutants and that Ca2+ weakly interacts with the S589D mutant. The block of alphaS589C, -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 gamma subunit in the background of the ENaC alphaS589C, -D, or -N mutants reduces the sensitivity to block by Cd2+ and renders the block rapidly reversible. We conclude therefore that the block by Cd2+ of the alphaS589C, -D, and -N mutants results from the trapping of Cd2+ ions in the internal pore of the channel and involves Cys-546 in the second transmembrane helix of the gammaENaC subunit.
Highlights
31928 JOURNAL OF BIOLOGICAL CHEMISTRY and is regulated by aldosterone [2]
The ENaC subunits belong to the ENaC/degenerin superfamily of ion channels but share no sequence homology with other tetrameric channels made of two transmembrane domains, such as inward rectifier Kϩ channels
In order to block ENaC, Cd2ϩ has to bind to distinct sites deep in the channel pore, including a cysteine residue in the TM2 of ␥ENaC subunits
Summary
31928 JOURNAL OF BIOLOGICAL CHEMISTRY and is regulated by aldosterone [2]. The aldosterone-sensitive distal nephron plays an important role in the maintenance of Naϩ homeostasis, the regulation of extracellular volume, and blood pressure [3]. ENaC and the Kϩ channel KcsA share a similar membrane topology and a heterotetrameric structure made of homologous subunits comprising two transmembrane segments. The amino-terminal portion of the pore region in the KcsA forms an ␣ helix (pore helix) pointing from the outside toward the center of the channel [12] Following this pore helix, the amino acid stretch lining the selectivity filter folds back toward the external surface, exposing binding sites for external blocking ligands such as toxins or triethanolamine (TEA). In ENaC, the pore region points from the outside to the center of the channel with an amino-to-carboxyl sense of, successively, the amiloride binding site, the selectivity filter, and the TM2 [1]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
