The Na, K-ATPase is the plasma membrane enzyme that actively transports Na+ and K+ against their electrochemical gradients. It is the single largest consumer of energy in the central nervous system, accounting for 40-50% of ATP hydrolysis. There are three genetically distinct isoforms of its catalytic (alpha) subunit, as well as two isoforms of its glycoprotein (beta) subunit. The fundamental role of the Na,K-ATPase is well-known, but we propose to ascertain its regulation and its participation in glial K+ clearance by investigating the isoforms expressed in different brain cells. The first hypothesis is that Na,K-ATPase beta subunits, like alpha subunits, have different cellular distributions in the nervous system. New isoform-specific monoclonal antibodies will be used to characterize the Na, K-ATPase beta subunits and localize them to identified cell types by immunocytochemistry on tissue sections. Antibodies will be extensively characterized, and specificity will be rigorously determined with a new molecular approach utilizing an M13 phage random peptide library. In our primary cultures of glia, we see an unprecedented number of differentiated cell types. The composition of mixed and separated cultures, and their Na,K-ATPase isoform gene expression, will be characterized and related to glial phenotype in vivo. We have new evidence suggesting that the alpha1 isoform of Na, K-ATPase has unusual functional characteristics in primary cultures of glia, and the second hypothesis is that the enzyme is altered to function better as a K+ pump. The biochemical properties of the membrane-bound na, K-ATPase will be determined, and Na,K-ATPase activity in intact glia will be investigated by ion transport and histochemistry. The Na,K-ATPase properties of different glial types will be characterized individually. We will determine whether the altered function involves alpha2, beta1, and beta2 by expression of cloned subunits. An exciting development is that a beta2 null mutant mouse dies before weaning with gross swelling of CNS astrocyte endfeet, demonstrating that beta2 is uniquely important for glial function. We will study glial survival and ATPase properties in cultures prepared from this transgenic mouse. Available evidence suggests that the isoforms differ in affinities for Na+, ATP and cardiac glycosides, and in susceptibility to regulation by unidentified intracellular factors. The third hypothesis is that the isoforms have different susceptibilities to regulation by protein kinases A and C. Our novel results predict that PKA and PKC actions will be affected oppositely by the physiological state of Na,K-ATPase (resting or turning-over). This will be critically evaluated with biochemical techniques, using purified enzyme and cultured cells.
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