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

摘要: Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, interact with lipid bilayers affect metabolic pathways. We previously that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium (ENaCs) in a cAMP-dependent manner involving stimulation of protein kinase (PKA). In the present study, we explored further mechanism EPA ENaC A6 cells. Fluorescence resonance energy transfer experiments confirmed activation PKA by EPA. Consistent our previous studies, had no stimulatory effect on amiloride-sensitive transepithelial current (INa) presence CPT-cAMP. Thus, investigated cellular pathways which produce cAMP. did not stimulate adenylate cyclase activity or total cAMP accumulation. However, membrane-bound phosphodiesterase was inhibited from 2.46 pmol/mg protein/min 1.3 protein/min. To investigate potential role an A-kinase-anchoring (AKAP), used HT31, inhibitor binding between AKAPs as well cerulenin, myristoylation palmitoylation. Both agents prevented CPT-cAMP INa drastically decreased amount apical membrane. Colocalization cells cotransfected fluorescently labeled β subunit regulatory close proximity two proteins membrane anchorage PKA. Last, transfected dead mutant Sgk, enzyme up-regulates ENaCs, Na+ current. Our results suggest ENaCs occurs via SGK compartments containing AKAP, activated PKA, phosphodiesterase.