MOLECULAR ANALYSIS OF NA+/K+ ATPASE

Project: Research project

Description

The objective of this proposal is the molecular analysis of the
expression, structure, and function of Na,K-ATPase isoforms expressed in
brain. Specific aims include: 1) Regulation of Na,K,ATPase gene
expression. We will attempt to characterize the DNA sequences and
cellular factors that promote cell-specific activation of the Na,K-ATPase
alpha3 and beta2 subunit genes in the CNS. We will first use gene
transfer methods to identify sequence elements within the alpha3 and
beta2 genes that promote efficient and cell-specific expression of a
reporter gene. We will also attempt to use retrovirus-mediated gene
transfer to introduce reporter gene constructs directly into the
postnatal rat retina. Because alpha3 and beta2 subunits are naturally
produced in photoreceptor cells, it should be possible to identify
regulatory elements within the alpha3 and beta2 genes that are required
to promote photoreceptor cell-specific gene expression. 2) Structure-
function analysis. We will attempt to identify sequences within the a
subunit responsible for the wide variation in sodium affinity between the
Hydra alpha subunit and rat alpha1 subunit. Construction and expression
of chimeras between Hydra alpha and rat alpha1 subunit cDNAs should
permit identification of sites that interact with Na+ and contribute to
Na+ binding. In order to develop a detailed topographical map of the a
subunit, we will use epitope addition to create a panel of alpha1 subunit
cDNAs carrying insertional tags. Indirect immunofluorescence microscopy
of transfectants with a monoclonal antibody reactive with the epitope tag
should permit us to determine whether an epitope-tagged domain is located
on the inside or outside of the cell. 3) Functional significance for
isoform diversity. The identification of cell types expressing limited
combinations of alpha and beta subunits makes it possible to compare the
biochemical properties of distinct Na,K-ATPase isoenzymes. Initial
interest will focus on a comparison of the substrate requirements of the
pineal gland alpha3/beta2-containing isoenzyme and the neuronal enzyme
composed of alpha3 and beta1 subunits. This approach should allow us to
begin to understand the functional differences between Na,K-ATPase
isoenzymes and determine whether the beta subunit contributes to the
affinity of a specific alpha subunit for Na+ and/or K+.
StatusFinished
Effective start/end date7/1/936/30/98

Funding

  • National Institutes of Health
  • National Institutes of Health: $21,202.00
  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $209,392.00

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Epitopes
Photoreceptor Cells
Isoenzymes
Hydra
Genes
Gene Expression
Reporter Genes
Retroviridae
Indirect Fluorescent Antibody Technique
Protein Isoforms
Complementary DNA
Retina
Sodium
Monoclonal Antibodies
sodium-translocating ATPase
Pineal Gland
Fluorescence Microscopy