The sodium pump has been the target for the therapeutic treatment of congestive heart failure with cardiac glycosides for more than a century. Unfortunately there is still little known about the mechanism of cardiac glycoside inhibition. Experimental studies over the past three decades have established some relationships between the biochemical reactions catalyzed by the sodium pump and the transport reactions it mediates. However, the molecular mechanisms by which the hydrolysis of ATP is coupled to the """"""""uphill"""""""" movement of ions remains a mystery. This probably stems from the fact that there remains little known about the structure-function relationship of this protein. Our long term goal is to gain a complete understanding of the sodium pump transport mechanism which will require high-resolution x-ray diffraction patterns of the enzyme in its various conformations and the identification of specific amino acids residues associated with ligand binding. However, the techniques required to get quality crystals of membrane proteins have not yet been perfected. The specific experiments outlined in this proposal will exploit bacterial genetics to overexpress the large cytoplasmic loop of the Na,K-ATPase (Aim I). All the residues thus far implicated in ATP binding and hydrolysis have been found in this section of the protein. The same holds true for all members of this important protein family (i.e., P-type ATPases). The ATP binding characteristics of the wild type and mutant ATP binding domains will be determined and structurally characterized using CD and x-ray crystallographic techniques (Aim III). In addition, we will determine which other sections of the Na,K-ATPase physically interact with the ATP binding domain (Aim II). The results from this work will provide a map of the amino acids involved in ATP coordination and thus help elucidate the coupling mechanism between ATP hydrolysis and cation transport. The sodium pump is vital in a variety of organs for fluid and electrolyte balance. These processes are in a dynamic equilibrium and this equilibrium can become disrupted in a variety of disease states. Before an adequate description of these pathological situations can be made, a more complete understanding of sodium pump function is required.
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