Chapter title |
Functional Studies of Na+,K+-ATPase Using Transfected Cell Cultures
|
---|---|
Chapter number | 28 |
Book title |
P-Type ATPases
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3179-8_28 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3178-1, 978-1-4939-3179-8
|
Authors |
Elena Arystarkhova, Kathleen J. Sweadner, Arystarkhova, Elena, Sweadner, Kathleen J. |
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. |
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