MOLECULAR ANALYSIS OF NA+/K+ ATPASE

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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+.