Project: Research project

Project Details


The goal of the proposed project is to isolate, characterize and
use as an analytical tool the DNA sequences coding for the Na,K-
ATPase alpha and beta subunits from rat heart. By comparing rat
heart cDNAs with the previously characterized alpha and beta
subunits genes from rat brain we will determine whether Na,K-
ATPase molecules of differing primary sequence are present in
heart tissue. The availability of cDNA probes for the Na,K-
ATPase will permit us to address basic issues with respect to
control mechanisms which affect myocardial Na,K-ATPase.
These include: a) Analysis of ATPase gene expression in specific
cells of the heart. We will use in situ hybridization techniques to
determine whether alternative forms of ATPase mRNA are
expressed in specific cells of the developing and adult rat heart.
b) Regulation of ATPase gene expression. By nucleic acid
hybridization techniques, we will determine whether physiological
stress can lead to regulation of the activity of the Na,K-ATPase
in a cell via alteration in the expression of Na,K-ATPase mRNA.
c) Organization of the ATPase Gene Family. In order to
understand the molecular basis for Na,K-ATPase isoform
diversity, we will attempt to determine the number of copies of
the ATPase gene in rat and examine the organization of these
genes. This approach could also lead to the identification of
regulatory regions which may control cell and tissue-specific
expression of the Na,K-ATPase gene family. d) Development of
Na,K-ATPase antibodies. Identification of sites of primary
sequence difference between ATPase isoforms will form the basis
for the development of a panel of antibodies capable of
recognizing specific isoforms of the Na,K-ATPase. These
antibodies will be used to gain insight into the anatomical location
of ATPase isoforms and the mechanisms underlying ATPase
biogenesis and subunit assembly. Introduction of the genes coding
for the alpha subunit of the ATPase into a mammalian cell will
allow us to study many aspects of the relationship between the
structure and function of the Na,K-ATPase. For example,
construction of chimeric cDNA molecules between ouabain-
resistant and ouabain sensitive forms of the ATPase should permit
us to define the region of the ATPase alpha subunit responsible
for differential ouabain sensitivity. This type of approach,
coupled with site specific mutagenesis, should make it feasible to
identify and study other functional domains in the Na,K-ATPase.
Effective start/end date7/1/874/30/97


  • National Institutes of Health
  • National Institutes of Health

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