title: The glycolytic enzymes glyceraldehyde 3-phosphate dehydrogenase and enolase interact with the renal epithelial K+ channel ROMK2 and regulate its function.
abstract: OBJECTIVE
  ROMK channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. The aim was to study the functional implications of the interaction between ROMK2 (Kir1.1b) and two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase-α, which were identified as potential regulatory subunits of the channel complex.
  
  
  METHODS
  We performed a membrane yeast-two-hybrid screen of a human kidney cDNA library with ROMK2 as a bait. Interaction of ROMK2 with GAPDH and enolase was verified using GST pull-down, co-immunoprecipitation, immunohistochemistry and co-expression in Xenopus oocytes.
  
  
  RESULTS
  Confocal imaging showed co-localisation of enolase and GAPDH with ROMK2 in the apical membrane of the renal epithelial cells of the thick ascending limb. Over-expression of GAPDH or enolase-α in Xenopus oocytes markedly reduced the amplitude of ROMK2 currents but did not affect the surface expression of the channels. Co-expression of the glycolytically inactive GAPDH mutant C149G did not have any effect on ROMK2 current amplitude.
  
  
  CONCLUSIONS
  Our results suggest that the glycolytic enzymes GAPDH and enolase are part of the ROMK2 channel supramolecular complex and may serve to couple salt reabsorption in the thick ascending limb of the loop of Henle to the metabolic status of the renal epithelial cells.
authors.affiliation: Department of Paediatric Nephrology, Children's Hospital, University of Marburg, Baldingerstrasse, Marburg, Germany.
authors.forename: Aparna
authors.initials: A
authors.lastname: Renigunta
authors.forename: Kerim
authors.initials: K
authors.lastname: Mutig
authors.forename: Kathrin
authors.initials: K
authors.lastname: Rottermann
authors.forename: Günter
authors.initials: G
authors.lastname: Schlichthörl
authors.forename: Regina
authors.initials: R
authors.lastname: Preisig-Müller
authors.forename: Jürgen
authors.initials: J
authors.lastname: Daut
authors.forename: Siegfried
authors.initials: S
authors.lastname: Waldegger
authors.forename: Vijay
authors.initials: V
authors.lastname: Renigunta
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation; D007668:Kidney / Q000201:enzymology / Q000378:metabolism; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism / Q000502:physiology*; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis / Q000235:genetics
chemical_list: D024661:Potassium Channels, Inwardly Rectifying; C571352:ROMK2 protein, human; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010751:Phosphopyruvate Hydratase