title: Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).
abstract: Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.
authors.affiliation: Department of Medicine, Division of Nephrology, The University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. hepingma@uab.edu
authors.forename: He-Ping
authors.initials: HP
authors.lastname: Ma
authors.forename: Sunil
authors.initials: S
authors.lastname: Saxena
authors.forename: David G
authors.initials: DG
authors.lastname: Warnock
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D004232:Diuretics / Q000494:pharmacology; D053503:Epithelial Sodium Channels; D005260:Female; D005786:Gene Expression Regulation; D007477:Ions; D018613:Microscopy, Confocal; D009399:Nephrons / Q000166:cytology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D011107:Polylysine / Q000737:chemistry; D018075:RNA, Complementary / Q000378:metabolism; D051381:Rats; D015222:Sodium Channels / Q000378:metabolism*; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism; D014981:Xenopus
chemical_list: D004232:Diuretics; D053503:Epithelial Sodium Channels; D007477:Ions; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010718:Phosphatidylserines; D010743:Phospholipids; D018075:RNA, Complementary; D015222:Sodium Channels; C060974:phosphatidylinositol 3,4,5-triphosphate; D011107:Polylysine; D000584:Amiloride; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases