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
| Autor: | Laurent Héliot, Sarah Sariban-Sohraby, Corentin Spriet, Frédérique Mies |
|---|---|
| Rok vydání: | 2007 |
| Předmět: |
A-kinase-anchoring protein
Biology Transfection Biochemistry Cell Line chemistry.chemical_compound Xenopus laevis Palmitoylation Fatty Acids Omega-3 Cyclic AMP Fluorescence Resonance Energy Transfer Animals Protein kinase A Epithelial Sodium Channels Molecular Biology Myristoylation Phosphoric Diester Hydrolases Cell Membrane Sodium Phosphodiesterase Cell Biology Apical membrane Cyclic AMP-Dependent Protein Kinases Cerulenin chemistry Microscopy Fluorescence Fatty Acids Unsaturated lipids (amino acids peptides and proteins) Cyclase activity Protein Binding |
| Zdroj: | The Journal of biological chemistry. 282(25) |
| ISSN: | 0021-9258 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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