title: Functional Studies of Na(+),K(+)-ATPase Using Transfected Cell Cultures.
abstract: The properties of different combinations of Na,K-ATPase subunits or their mutations can be studied in stably transfected mammalian cells. As a specific example, the methods here are for transfection of a modulatory subunit into cells with endogenous α and β subunits. Renal Na,K-ATPase is tightly bound to a small single-span membrane protein, the γ subunit, or FXYD2. The protein co-localizes and co-immunoprecipitates with the α/β complex, however it is not required for basic enzyme properties. Functional consequences of association with FXYD2 were investigated in stably transfected cells. The outcome was that FXYD2 reduced activity of Na,K-ATPase at the level of apparent affinity for Na(+) and to a smaller extent for K(+). Moreover, expression of FXYD2 reduced cell growth. Here we describe the methodologies as well as potential pitfalls.
authors.affiliation: Laboratory of Membrane Biology, Massachusetts General Hospital, Edwards 410, 55 Fruit St., Boston, MA, 02114, USA. aristarkhova@helix.mgh.harvard.edu.
authors.forename: Elena
authors.initials: E
authors.lastname: Arystarkhova
authors.affiliation: Laboratory of Membrane Biology, Massachusetts General Hospital, Edwards 410, 55 Fruit St., Boston, MA, 02114, USA.
authors.forename: Kathleen J
authors.initials: KJ
authors.lastname: Sweadner
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation; D003001:Cloning, Molecular; D006801:Humans; D010042:Ouabain / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D010042:Ouabain; D000255:Adenosine Triphosphate; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium
keywords: Affinity; Cell growth; FXYD; Gamma subunit; Na,K-ATPase activity; Stable transfectants