MOLECULAR PHYSIOLOGY OF THE NAK-ATPASE

Washington University, Saint Louis MO

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Abstract

The Na,K-ATPase is an important membrane-associated enzyme responsible for maintaining the high internal potassium concentration and low internal sodium concentration characteristic of most animal cells. The ion gradients created by the Na,K-ATPase are fundamental to such diverse cellular functions as the regulation of cell volume and pH, the uptake of nutrients and diverse cellular functions as the regulation of cell volume and pH, the uptake of nutrients and membrane excitability. Because of its pivotal role in ion and water homeostasis, alternations in Na,K-ATPase activity are associated with several cardiovascular, renal and neurological disorders. The present studies will use the techniques of molecular physiology to study the structure, function and regulation of the Na,K-ATPase. This will involve: 1) The identification and characterization of amino acids located within the binding sites for Na+, K+ and ATP. This will be accomplished by modifying alpha subunit amino acids and evaluating the effects these replacements have on cation and nucleotide binding. In addition, PCR-based random mutagenesis will be used to identify amino acids important in K+ binding. 2) To determine the region(s) of the Na,K-ATPase beta subunit that influences cation affinity. 3) To determine if palytoxin induced ion fluxes utilize the normal pathway for ions through the Na,K-ATPase. 4) To identify the specific amino acids within the alpha subunit that are required for oligomerization and to ascertain if a subunit oligomerization affects Na,K-ATPase function. Finally, 5) To further characterize the effect of regulatory phosphorylation of the alpha subunit isoforms on Na,K-ATPase function. The proposed studies will contribute to a better understanding of this important transport protein.