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J. Biol. Chem., Vol. 280, Issue 19, 19003-19011, May 13, 2005
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Na,K-ATPase from Mice Lacking the 
Subunit (FXYD2) Exhibits Altered Na+ Affinity and Decreased Thermal Stability*
From the aDepartments of Physiology and Pharmacology, Obstetrics and Gynaecology, and Paediatrics, the University of Western Ontario, London, Ontario N6A 5C1, Canada and Children's Health Research Institute, London, Ontario N6C 2V5, Canada, the dLaboratory of Membrane Biology, Massachusetts General Hospital, Boston, Massachusetts 02114 and Harvard Medical School, Boston, Massachusetts 02115, and fThe Lawson Research Institute, St. Joseph's Health Centre, London, Ontario N6A 1Y5, Canada
The 
subunit of the Na,K-ATPase, a 7-kDa single-span membrane protein, is a member of the FXYD gene family. Several FXYD proteins have been shown to bind to Na,K-ATPase and modulate its properties, and each FXYD protein appears to alter enzyme kinetics differently. Different results have sometimes been obtained with different experimental systems, however. To test for effects of in a native tissue environment, mice lacking a functional subunit gene (Fxyd2) were generated. These mice were viable and without observable pathology. Prior work in the mouse embryo showed that is expressed at the blastocyst stage. However, there was no delay in blastocele formation, and the expected Mendelian ratios of offspring were obtained even with Fxyd2–/– dams. In adult Fxyd2–/– mouse kidney, splice variants of that have different nephron segment-specific expression patterns were absent. Purified -deficient renal Na,K-ATPase displayed higher apparent affinity for Na+ without significant change in apparent affinity for K+. Affinity for ATP, which was expected to be decreased, was instead slightly increased. The results suggest that regulation of Na+ sensitivity is a major functional role for this protein, whereas regulation of ATP affinity may be context-specific. Most importantly, this implies that and other FXYD proteins have their effects by local and not global conformation change. Na,K-ATPase lacking the subunit had increased thermal lability. Combined with other evidence that participates in an early step of thermal denaturation, this indicates that FXYD proteins may play an important structural role in the enzyme complex.
Received for publication, January 19, 2005 , and in revised form, March 7, 2005.
* This work was supported by grants from the Medical Research Council of Canada (to G. M. K. and G.-H. F.), the Canadian Institutes of Health Research (to G. M. K.), and the NHLBI Grant R01-HL36271 from the National Institutes of Health (to K. J. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b These authors contributed equally to this work.
c Present address: University of Ontario Institute of Technology, 2000 Simcoe Street N., Oshawa, Ontario L1H 7L7, Canada.
e Present address: Cell Signalling Technology, 166B Cummings Center, Beverly, MA 01915.
g Present address: Van Andel Research Institute, 333 Bostwick Ave. N.E., Grand Rapids, MI 49503.
h Present address: Genes and Development Research Group, Dept. of Biochemistry and Molecular Biology, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alberta T2N 4N1, Canada.
i Present address: Dept. of Physiology/Center for Vascular Biology, University of Connecticut Health Center, 263 Farmington Ave., Farmington, CT 06030.
j To whom correspondence should be addressed: Dept. of Physiology and Pharmacology, Dental Sciences Bldg., Dock 15, the University of Western Ontario, London, Ontario N6A 5C1, Canada. Tel.: 519-661-3132; Fax: 519-850-2562; E-mail: gerald.kidder{at}fmd.uwo.ca.
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