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.

Details

Ressource 1Download: BIB_969CF4BDB749.P001.pdf (264.66 [Ko])
State: Public
Version: author
Serval ID
serval:BIB_969CF4BDB749
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
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.
Journal
Journal of General Physiology
Author(s)
Schild L., Schneeberger E., Gautschi I., Firsov D.
ISSN
0022-1295 (Print)
ISSN-L
0022-1295
Publication state
Published
Issued date
1997
Volume
109
Number
1
Pages
15-26
Language
english
Abstract
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.
Keywords
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
Yes
Create date
24/01/2008 12:32
Last modification date
20/08/2019 14:58
Usage data