The objective of this proposal is to analyze the relationship between the structure and function of Na,K-ATPase.
The specific aims are: 1. Na,K-ATPase alpha/beta subunit interaction. The applicant will attempt to determine which combinations of alpha and beta subunit isoforms can associate by inserting epitope tags into cDNAs encoding alpha and beta subunit isoforms and expressing the constructs in mammalian cells. 2. Structure-function of the alpha subunit. The applicant will develop a refined topographical map of the alpha subunit polypeptide by creating a cysteine-less alpha subunit mutant. Cysteine residues will be sequential introduced into predicted extracellular or cytoplasmic loops of the cysteine-less mutant, and the topology of individual cysteine tags determined using biotin-labeled thiol- or sulfhydryl-reactive reagents. A second goal will be to identify sequences within the alpha subunit that form the sodium binding site. 3. Genetic analysis of Looptail mice:Function of the alpha2 subunit. The applicant will attempt to determine whether Looptail (Lp) mice exhibit alterations in the sequence or expression of the Na,K-ATPase alpha2 subunit (Atp1a2) gene. 4. Molecular analysis of the beta3 subunit. The applicant proposes to further characterize the recently discovered Na,K-ATPase beta3 subunit. The goal is to isolate and characterize cDNA and genomic DNA sequences for the murine beta3 subunit and develop antibody probes for cell localization studies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL039263-14
Application #
6056198
Study Section
Cardiovascular and Pulmonary Research A Study Section (CVA)
Project Start
1987-07-01
Project End
2001-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
14
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Pennsylvania State University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
129348186
City
Hershey
State
PA
Country
United States
Zip Code
17033
Cheng, Keith C; Levenson, Robert; Robishaw, Janet D (2003) Functional genomic dissection of multimeric protein families in zebrafish. Dev Dyn 228:555-67
Rajarao, Johannes R; Canfield, Victor A; Loppin, Benjamin et al. (2002) Two Na,K-ATPase beta 2 subunit isoforms are differentially expressed within the central nervous system and sensory organs during zebrafish embryogenesis. Dev Dyn 223:254-61
Rajarao, S J; Canfield, V A; Mohideen, M A et al. (2001) The repertoire of Na,K-ATPase alpha and beta subunit genes expressed in the zebrafish, Danio rerio. Genome Res 11:1211-20
Dahl, J P; Binda, A; Canfield, V A et al. (2000) Participation of Na,K-ATPase in FGF-2 secretion: rescue of ouabain-inhibitable FGF-2 secretion by ouabain-resistant Na,K-ATPase alpha subunits. Biochemistry 39:14877-83
Underhill, D A; Canfield, V A; Dahl, J P et al. (1999) The Na,K-ATPase alpha4 gene (Atp1a4) encodes a ouabain-resistant alpha subunit and is tightly linked to the alpha2 gene (Atp1a2) on mouse chromosome 1. Biochemistry 38:14746-51
Malik, N; Canfield, V; Sanchez-Watts, G et al. (1998) Structural organization and chromosomal localization of the human Na,K-ATPase beta 3 subunit gene and pseudogene. Mamm Genome 9:136-43
Canfield, V A; Levenson, R (1998) Domain swapping between Na,K- and H,K-ATPase identifies regions that specify Na,K-ATPase activity. Biochemistry 37:7509-16
Billecocq, A; Horne, W C; Chakraborty, M et al. (1997) 1,25-Dihydroxyvitamin D3 selectively induces increased expression of the Na,K-ATPase beta 1 subunit in avian myelomonocytic cells without a concomitant change in Na,K-ATPase activity. J Cell Physiol 172:221-9
Canfield, V A; Norbeck, L; Levenson, R (1996) Localization of cytoplasmic and extracellular domains of Na,K-ATPase by epitope tag insertion. Biochemistry 35:14165-72
Malik, N; Canfield, V A; Beckers, M C et al. (1996) Identification of the mammalian Na,K-ATPase 3 subunit. J Biol Chem 271:22754-8

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