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.
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