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
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.
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