Epithelial Na+ channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase.
Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/min to 1.3 pmol/mg of protein/min. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC beta subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase.
Mies
Frédérique
F
Physiology Department, Université Libre de Bruxelles, 808 Route de Lennik, CP604, 1070 Belgium.
Spriet
Corentin
C
Biophotonique Cellulaire Fonctionnelle, Interdisciplinary Research Institute, 1 rue du Prof. Calmette, BP447, 59021 Lille Cedex, France.
Héliot
Laurent
L
Biophotonique Cellulaire Fonctionnelle, Interdisciplinary Research Institute, 1 rue du Prof. Calmette, BP447, 59021 Lille Cedex, France.
Sariban-Sohraby
Sarah
S
Physiology Department, Université Libre de Bruxelles, 808 Route de Lennik, CP604, 1070 Belgium. Electronic address: sohrabys@nei.nih.gov.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D015525:Fatty Acids, Omega-3 / Q000378:metabolism*; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D008856:Microscopy, Fluorescence; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D012964:Sodium / Q000378:metabolism; D014162:Transfection; D014982:Xenopus laevis
D053503:Epithelial Sodium Channels; D015525:Fatty Acids, Omega-3; D005231:Fatty Acids, Unsaturated; D012964:Sodium; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases