Functional Studies of Na(+),K(+)-ATPase Using Transfected Cell Cultures

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.