Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

Détails

ID Serval
serval:BIB_1F8DCC9E1FDA
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.
Périodique
Proceedings of the National Academy of Sciences of the United States of America
Auteur(s)
Firsov D., Schild L., Gautschi I., Mérillat A.M., Schneeberger E., Rossier B.C.
ISSN
0027-8424 (Print)
ISSN-L
0027-8424
Statut éditorial
Publié
Date de publication
1996
Volume
93
Numéro
26
Pages
15370-15375
Langue
anglais
Résumé
The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.
Mots-clé
Amino Acid Sequence, Animals, Antibodies, Monoclonal, Cell Membrane/physiology, Epithelium/metabolism, Epitopes, Female, Humans, Hypertension/genetics, Hypertension/metabolism, Kinetics, Molecular Sequence Data, Oocytes/physiology, Peptides, Protein Structure, Secondary, Rats, Sequence Homology, Amino Acid, Sodium Channels/analysis, Sodium Channels/chemistry, Syndrome, 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 13:55
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