The Na,K-ATPase (a P-type ion pump) catalyzes active transport of Na+ and K+ in almost all animal cells. It is essential for transepithelial transport, such as in the kidney, and for the ion gradients that support electrical excitability in muscle and nerve. It is the receptor for inotropic cardiac glycosides in the heart, and many studies point to a role in hypertension. Its catalytic subunit belongs to a family of ion transport ATPases. The crystal structure of the first of these, the Ca2+ ATP of sarcoplasmic reticulum, has recently been determined, providing new insight into the possible mechanism of ion transport. Our theoretical analysis of Na,K-ATPase aligned with Ca2+-ATPase suggests that certain past assumptions about Na,K-ATPase structure were wrong, and that the Ca2+-ATPase structure provides a workable framework for testing specific hypotheses about Na,K-ATPase structure. This proposal will test the hypothesis that the Na,K-ATPase alpha subunit adopts the same fold as the Ca2+-ATPase, using monoclonal antibodies with mapped epitopes as structural probes. Antibodies against the Na,K-ATPase extracellular surface will be produced to facilitate purification and eventual crystallization of the Na,K ATPase. We will also focus our attention on the two Na,K-ATPase subunits that have no counterpart in the Ca2+ - ATPase: the beta and gamma subunits. We will test specific hypotheses about where and how these subunits associate with the alpha subunit, using cross-linking of native and mutagenized enzyme. We have found that the gamma subunit modulates the most physiologically significant properties of the Na,K-ATPase, its affinity for Na+ and K+, and we will exploit that to map the essential structural features of gamma by saturation mutagenesis. We will test the hypothesis that regulation by gamma, like the similar but distinct phospholamban protein that modulates the Ca2+-ATPase, entails reversible oligomerization in the membrane. In sum, a combination of protein chemistry, hybridoma technology, and molecular approaches will be used to investigate the structure of an essential plasma membrane protein.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL036271-17
Application #
6638259
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Buxton, Denis B
Project Start
1987-07-01
Project End
2005-06-30
Budget Start
2003-07-01
Budget End
2004-06-30
Support Year
17
Fiscal Year
2003
Total Cost
$346,000
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Arystarkhova, Elena; Sweadner, Kathleen J (2016) Functional Studies of Na(+),K(+)-ATPase Using Transfected Cell Cultures. Methods Mol Biol 1377:321-32
Sweadner, Kathleen J (2016) Colorimetric Assays of Na,K-ATPase. Methods Mol Biol 1377:89-104
Arystarkhova, Elena; Liu, Yi B; Salazar, Cynthia et al. (2013) Hyperplasia of pancreatic beta cells and improved glucose tolerance in mice deficient in the FXYD2 subunit of Na,K-ATPase. J Biol Chem 288:7077-85
Sweadner, Kathleen J; Pascoa, Jennifer L; Salazar, Cynthia A et al. (2011) Post-transcriptional control of Na,K-ATPase activity and cell growth by a splice variant of FXYD2 protein with modified mRNA. J Biol Chem 286:18290-300
Arystarkhova, Elena; Donnet, Claudia; Munoz-Matta, Ana et al. (2007) Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-subunit in response to stress. Am J Physiol Cell Physiol 292:C1179-91
Jia, Li-Guo; Donnet, Claudia; Bogaev, Roberta C et al. (2005) Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice. Am J Physiol Heart Circ Physiol 288:H1982-8
Arystarkhova, Elena; Sweadner, Kathleen J (2005) Splice variants of the gamma subunit (FXYD2) and their significance in regulation of the Na, K-ATPase in kidney. J Bioenerg Biomembr 37:381-6
Jones, D Holstead; Li, Tony Y; Arystarkhova, Elena et al. (2005) Na,K-ATPase from mice lacking the gamma subunit (FXYD2) exhibits altered Na+ affinity and decreased thermal stability. J Biol Chem 280:19003-11
Sweadner, Kathleen J (2005) Phospholemman: a new force in cardiac contractility. Circ Res 97:510-1
Wetzel, Randall K; Pascoa, Jennifer L; Arystarkhova, Elena (2004) Stress-induced expression of the gamma subunit (FXYD2) modulates Na,K-ATPase activity and cell growth. J Biol Chem 279:41750-7

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