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J. Biol. Chem., Vol. 282, Issue 25, 18339-18347, June 22, 2007

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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^*

Frédérique Mies, Corentin Spriet¹, Laurent Héliot, and Sarah Sariban-Sohraby²

From the Physiology Department, Université Libre de Bruxelles, 808 Route de Lennik, CP604, 1070 Belgium and the Biophotonique Cellulaire Fonctionnelle, Interdisciplinary Research Institute, 1 rue du Prof. Calmette, BP447, 59021 Lille Cedex, France

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 (I_Na) 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 I_Na and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC 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.

Received for publication, December 5, 2006 , and in revised form, March 29, 2007.

^* This work was supported by funds from Université Libre de Bruxelles and from the Fonds National pour la Recherche Scientifique (FNRS), the ANR-05-MIIM-042, and the Mission des Ressources et Compétences Technologiques. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by Region-Nord-Pas-de-Calais, ANR-05-MIIM-042, and MRCT.

² To whom correspondence should be addressed: National Institutes of Health, Bldg. 31, Rm. 6A22, Bethesda, MD 20892. Tel.: 301-451-6763; Fax: 301-451-6764; E-mail: sohrabys{at}nei.nih.gov.

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