title: Carbonic anhydrase II binds to and increases the activity of the epithelial sodium-proton exchanger, NHE3.
abstract: Two-thirds of sodium filtered by the renal glomerulus is reabsorbed from the proximal tubule via a sodium/proton exchanger isoform 3 (NHE3)-dependent mechanism. Since sodium and bicarbonate reabsorption are coupled, we postulated that the molecules involved in their reabsorption [NHE3 and carbonic anhydrase II (CAII)] might physically and functionally interact. Consistent with this, CAII and NHE3 were closely associated in a renal proximal tubular cell culture model as revealed by a proximity ligation assay. Direct physical interaction was confirmed in solid-phase binding assays with immobilized CAII and C-terminal NHE3 glutathione-S-transferase fusion constructs. To assess the effect of CAII on NHE3 function, we expressed NHE3 in a proximal tubule cell line and measured NHE3 activity as the rate of intracellular pH recovery, following an acid load. NHE3-expressing cells had a significantly greater rate of intracellular pH recovery than controls. Inhibition of endogenous CAII activity with acetazolamide significantly decreased NHE3 activity, indicating that CAII activates NHE3. To ascertain whether CAII binding per se activates NHE3, we expressed NHE3 with wild-type CAII, a catalytically inactive CAII mutant (CAII-V143Y), or a mutant unable to bind other transporters (CAII-HEX). NHE3 activity increased upon wild-type CAII coexpression, but not in the presence of the CAII V143Y or HEX mutant. Together these studies support an association between CAII and NHE3 that alters the transporter's activity.
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada; and.
authors.forename: Devishree
authors.initials: D
authors.lastname: Krishnan
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada;
authors.forename: Lei
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada; and.
authors.forename: Shane A
authors.initials: SA
authors.lastname: Wiebe
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada; Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada; and.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Casey
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada; and.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Cordat
authors.affiliation: Department of Physiology, University of Alberta, Edmonton, Alberta, Canada; Membrane Protein Disease Research Group, University of Alberta, Edmonton, Alberta, Canada; and Department of Pediatrics, University of Alberta, Edmonton, Alberta, Canada todd2@ualberta.ca.
authors.forename: R Todd
authors.initials: RT
authors.lastname: Alexander
mesh_terms: D000818:Animals; D001639:Bicarbonates / Q000378:metabolism*; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002460:Cell Line; D006801:Humans; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D009154:Mutation; D009893:Opossums; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D011994:Recombinant Proteins / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D011994:Recombinant Proteins; C000613385:SLC9A3 protein, human; D000074083:Sodium-Hydrogen Exchanger 3; D017923:Sodium-Hydrogen Exchangers; D012964:Sodium; D024402:Carbonic Anhydrase II
keywords: NHE3; carbonic anhydrase II; pH; sodium