NA-K-ATPASE ISOZYME LOCALIZATION AND FUNCTION IN CNS

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The Na,K-ATPase is the plasma membrane enzyme that actively transports Na + and K+ ions against their electrochemical gradients, with the hydrolysis of ATP. It is thought to be the single largest consumer of energy in the central nervous system, accounting for 30-40% of ATP hydrolysis. Three different isozymes of the Na,K-ATPase are expressed in the CNS, and in some instances are rather strikingly localized within different cell types. Their individual properties and physiological roles are not yet well known. Available evidence suggests that the isozymes may differ in their affinities for ATP and cardiac glycosides, and in their susceptibility to regulation by unidentified intracellular factors. In addition, ischemic injury has been reported to cause a selective and rapid inactivation of one of two isozymes in the heart. The hypothesis of this proposal is that the Na,K-ATPase isozymes in the CNS are expressed in different cell types, where their affinity for ATP or susceptibility to regulation or degradation may either exacerbate or ameliorate ischemic injury and edema. The properties of the Na,K-ATPases may play a role in explaining the different vulnerability to ischemia of different classes of neurons. The proposal has several objectives: to determine which cell types express which isozymes; to investigate their functional properties and predict which isozymes will remain active in marginally perfused tissue; and to investigate their inactivation secondary to a rise in intracellular Ca2+. We will utilize immunocytochemistry with specific monoclonal antibodies to determine the cellular and subcellular distribution of the Na,K-ATPases in the rat CNS. We will separate the isozymes, and determine their individual affinities for ATP, Na and K+, as well as investigate the possible use of GTP as a substrate. Finally we will investigate the mechanism of modification or inactivation of each isozyme by Ca 2+-dependent proteolysis and by calnaktin-mediated Ca 2+ inhibition. This basic research should lay the groundwork for a rigorous analysis of ion movements, energy consumption, and their role in cell death and edema in ischemic brain, and contribute to a detailed understanding of ion pump function.