title: Ezrin is required for the functional regulation of the epithelial sodium proton exchanger, NHE3.
abstract: The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd)) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.
authors: Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka, Japan.
authors: Hisayoshi
authors: H
authors: Hayashi
authors: Atsushi
authors: A
authors: Tamura
authors: Devishree
authors: D
authors: Krishnan
authors: Sachiko
authors: S
authors: Tsukita
authors: Yuichi
authors: Y
authors: Suzuki
authors: Hetal S
authors: HS
authors: Kocinsky
authors: Peter S
authors: PS
authors: Aronson
authors: John
authors: J
authors: Orlowski
authors: Sergio
authors: S
authors: Grinstein
authors: R Todd
authors: RT
authors: Alexander
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D003106:Colon / Q000187:drug effects / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D061985:Madin Darby Canine Kidney Cells; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008840:Microfilament Proteins / Q000378:metabolism*; D008871:Microvilli; D017830:Octoxynol / Q000737:chemistry; D010766:Phosphorylation; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000099149:Ezrin
chemical_list: D000242:Cyclic AMP; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D017830:Octoxynol; D034741:RNA, Small Interfering; D012964:Sodium; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D000099149:Ezrin; C000613385:SLC9A3 protein, human; C000613386:Slc9a3 protein, mouse; C071058:moesin; C071776:radixin