The epithelial sodium channel (ENaC) traffics to apical membrane in lipid rafts in mouse cortical collecting duct cells.
We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.
Hill
Warren G
WG
Division of Matrix Biology, Department of Medicine, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, MA 02215, USA.
Butterworth
Michael B
MB
Wang
Huamin
H
Edinger
Robert S
RS
Lebowitz
Jonathan
J
Peters
Kathryn W
KW
Frizzell
Raymond A
RA
Johnson
John P
JP
D000818:Animals; D022461:Caveolins / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D003505:Cyclodextrins / Q000737:chemistry; D003513:Cycloheximide / Q000737:chemistry; D053503:Epithelial Sodium Channels / Q000737:chemistry / Q000502:physiology*; D005799:Genes, Dominant; D049452:Green Fluorescent Proteins / Q000737:chemistry; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D020033:Protein Isoforms; D021381:Protein Transport
D022461:Caveolins; D003505:Cyclodextrins; D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D005576:Colforsin; D003513:Cycloheximide