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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Acknowledgements
This work was supported by NIH grants HL036271, NS045283, EY014390, NS050696, and NS081558.
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Arystarkhova, E., Sweadner, K.J. (2016). Functional Studies of Na+,K+-ATPase Using Transfected Cell Cultures. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_28
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DOI: https://doi.org/10.1007/978-1-4939-3179-8_28
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
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