The Carboxyl Terminus of the α-Subunit of the Amiloride-sensitive Epithelial Sodium Channel Binds to F-actin

James K. Bubien,Dale J. Benos,Christopher Mazzochi,Peter R. Smith

doi:10.1074/jbc.m509386200

James K. Bubien, Dale J. Benos + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m509386200

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Abstract

The activity of the amiloride-sensitive epithelial sodium channel (ENaC) is modulated by F-actin. However, it is unknown if there is a direct interaction between alpha-ENaC and actin. We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of alpha-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies. Confocal microscopy of Madin-Darby canine kidney cell monolayers stably transfected with wild type, rat isoforms of alpha-, beta-, and gamma-ENaC revealed co-localization of alpha-ENaC with the cortical F-actin cytoskeleton both at the apical membrane and within the subapical cytoplasm. F-actin was found to co-immunoprecipitate with alpha-ENaC from whole cell lysates of this cell line. Gel overlay assays demonstrated that F-actin specifically binds to the carboxyl terminus of alpha-ENaC. A direct interaction between F-actin and the COOH terminus of alpha-ENaC was further corroborated by F-actin co-sedimentation studies. This is the first study to report a direct and specific biochemical interaction between F-actin and ENaC.

Highlights

The amiloride-sensitive epithelial sodium channel (ENaC)2 is a member of the degenerin/epithelial sodium channel superfamily of ion channels
We have investigated the hypothesis that the actin cytoskeleton directly binds to the carboxyl terminus of ␣-ENaC using a combination of confocal microscopy, co-immunoprecipitation, and protein binding studies
A great deal of information is known about the biophysical properties of ENaC once it is inserted into the apical surface of an epithelial cell plasma membrane

Summary

Introduction

The amiloride-sensitive epithelial sodium channel (ENaC)2 is a member of the degenerin/epithelial sodium channel superfamily of ion channels. Co-localization of actin and ␣-ENaC in the apical membrane of MDCK cells stably expressing functional ENaC was demonstrated using laser-scanning confocal microscopy.

Results

Conclusion