NA K ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

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DESCRIPTION: This is a proposal to continue studies of Na,K-ATPase expression and function in the central nervous system (CNS). This enzyme controls ion gradients across plasma membranes, as well as the composition and volume of extracellular fluid. It consumes 50% of energy used in brain and 70% in retina. Since its several isoforms differ in substrate affinities and susceptibility to regulation, defining patterns of isoform expression is important to understanding roles of the enzyme in the CNS. This laboratory has developed antibodies specific for three isoforms of the Na,K-ATPase alpha subunit as well as for three beta subunit isoforms, and used these for localization studies in cerebellum and in cell culture. A third gamma subunit has been little studied. It is suggested that progress on the Human Genome Project will rapidly lead to identification of all subunit isoforms of this enzyme, as well as the related H,K-ATPases. In this context, the first aim of this proposal is to produce isoform-specific antibodies to all of the subunits expressed in the CNS, to determine their distributions by immunocytochemistry and confocal microscopy in brain, retina, cultured astrocytes, optic nerve and secretory structures such as choroid plexus and ciliary body. cDNAs for new subunits will be sequenced and characterized. The second aim will investigate functional properties of different isoform combinations in mammalian cells and transfectants to answer questions about their physiological roles and to determine the molecular basis for astrocyte potassium transport properties. The third aim is to use a histochemical assay for Na,K-ATPase activity to detect activity of different isoforms in situ, and to test the hypothesis that reduced enzyme activity after ischemia is cell- and isoform-selective and important as a neuroprotective mechanism. These aims will be accomplished using a combination of cellular, molecular and immunological methodologies.