The carboxyl terminus of the alpha-subunit of the amiloride-sensitive epithelial sodium channel binds to F-actin.
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.
Mazzochi
Christopher
C
Department of Physiology and Biophysics and Department of Cell Biology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Bubien
James K
JK
Smith
Peter R
PR
Benos
Dale J
DJ
D000199:Actins / Q000378:metabolism*; D000584:Amiloride / Q000737:chemistry*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004285:Dogs; D053503:Epithelial Sodium Channels; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D026941:Sodium Channel Blockers / Q000737:chemistry; D015222:Sodium Channels / Q000235:genetics / Q000378:metabolism* / Q000502:physiology
D000199:Actins; D053503:Epithelial Sodium Channels; D010446:Peptide Fragments; D021122:Protein Subunits; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D000584:Amiloride