Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.

Détails

Ressource 1Télécharger: BIB_969CF4BDB749.P001.pdf (264.66 [Ko])
Etat: Public
Version: de l'auteur
ID Serval
serval:BIB_969CF4BDB749
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Identification of amino acid residues in the alpha, beta, and gamma subunits of the epithelial sodium channel (ENaC) involved in amiloride block and ion permeation.
Périodique
Journal of General Physiology
Auteur(s)
Schild L., Schneeberger E., Gautschi I., Firsov D.
ISSN
0022-1295 (Print)
ISSN-L
0022-1295
Statut éditorial
Publié
Date de publication
1997
Volume
109
Numéro
1
Pages
15-26
Langue
anglais
Résumé
The amiloride-sensitive epithelial Na channel (ENaC) is a heteromultimeric channel made of three alpha beta gamma subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in beta and gamma subunits at position beta G525 and gamma G537 increased the apparent inhibitory constant (Ki) for amiloride by > 1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the alpha subunit increased amiloride Ki by 20-fold, without changing channel conducting properties. Coexpression of these mutated alpha beta gamma subunits resulted in a non-conducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by external Zn2+ ions, in particular the alpha S583C mutant showing a Ki for Zn2+ of 29 microM. Mutations of residues alpha W582L, or beta G522D also increased amiloride Ki, the later mutation generating a Ca2+ blocking site located 15% within the membrane electric field. These experiments provide strong evidence that alpha beta gamma ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of alpha beta gamma subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ ions at an external Na+ binding site preventing ion permeation through the channel pore.
Mots-clé
Amiloride/pharmacology, Amino Acid Sequence, Amino Acids/physiology, Animals, Cations, Divalent/pharmacology, Epithelium/metabolism, Female, Ions, Oocytes/metabolism, Permeability/drug effects, Rats, Sodium Channels/drug effects, Sodium Channels/genetics, Xenopus
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 13:32
Dernière modification de la notice
20/08/2019 15:58
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