MOLECULAR ANALYSIS OF NA+/K+-ATPASE

Project: Research project

Project Details

Description

The objective of this proposal is to apply the techniques of
molecular genetics to the analysis of Na,K-ATPase structure,
function and biogenesis. We have developed an expression system to
test the biological activity of cloned rodent Na,K-ATPase genes.
This system forms the framework for experiments designed to analyze
structure function relationships for the ATPase and control
mechanisms underlying ATPase biosynthesis and assembly. The
specific aims of the proposal include: 1) Interaction of the Na,K-
ATPase with Cardiac Glycosides. We will evaluate the relationship
between enzyme structure and the binding of cardiac glycosides.
Initial experiments will involve the construction of chimeric cDNA
molecules between cDNA molecules encoding ouabain resistant and
ouabain sensitive forms of the Na,K-ATPase in order to delineate
regions of the alpha chain which interact with the drug. We will
then use site directed mutagenesis to alter specific residues in
a cDNA encoding a ouabain sensitive ATPase in an effort to convert
the encoded enzyme to ouabain resistance. 2) Subcellular
Localization of Na,K-ATPase Isoforms. The technique of in situ
hybridization will be used to learn whether alternative forms of
Na,K-ATPase mRNA are expressed within the same or different cells
of a particular tissue. Peptide derived antibodies specific for
each ATPase isoform will be developed and used to distinguish
alternative ATPase isoforms in cells synthesizing more than one
isoform and to identify sites of subcellular localization. The goal
of these experiments will be to obtain basic information regarding
the relationship between isoform expression and function. 3)
Control Mechanisms Affecting Na,K-ATPase Biosynthesis. We will
attempt to isolate and characterize DNA sequences which may be
responsible for the tissue specific and developmentally regulated
expression of Na,K-ATPase mRNAs. Initial interest will focus on
identification of the promoter and other 5' control regions
involved in the regulation of Na,K-ATPase mRNA expression. We will
also attempt to identify regions within alpha and beta subunit
mRNAs which may play a role in the postranscriptional regulation
of subunit biosynthesis. 4) Relationship of the beta Subunit to
Na,K-ATPase Function. We will attempt to develop an assay system
which measures the biological activity of the 8 subunit by creating
a system in which overexpression of the beta subunit is required
for cell viability. This system will then be used to assess
structure function relationships for the beta subunit.
StatusFinished
Effective start/end date12/1/841/31/93

Funding

  • National Institutes of Health

Fingerprint Explore the research topics touched on by this project. These labels are generated based on the underlying awards/grants. Together they form a unique fingerprint.