title: Modulation of Na,K-ATPase by the gamma subunit: studies with transfected cells and transmembrane mimetic peptides.
abstract: The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the gamma subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4.
authors.forename: Athina
authors.initials: A
authors.lastname: Zouzoulas
authors.forename: Alex G
authors.initials: AG
authors.lastname: Therien
authors.forename: Rosemarie
authors.initials: R
authors.lastname: Scanzano
authors.forename: Charles M
authors.initials: CM
authors.lastname: Deber
authors.forename: Rhoda
authors.initials: R
authors.lastname: Blostein
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000737:chemistry; D019921:Biotinylation; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry*; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary; D051381:Rats; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D014162:Transfection
chemical_list: D010455:Peptides; D000255:Adenosine Triphosphate; D001120:Arginine; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D008274:Magnesium; D005998:Glycine