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.
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