The sodium pump expels sodium ions from cells. The resulting electrochemical potential gradient is coupled to a variety of important physiological processes, including cell volume regulation, transport of metabolites into cells, and the electrical excitation of nerve and muscle cells. Sodium pump is the immediate target of digitalis, a drug used to treat congestive heart failure. The calcium pump, which functions in muscle contraction, and the gastric proton pump, which is the target of a new class of antisecretory drugs, are related molecules that are important in human physiology. The long-term goal of the research program is to learn how ions are actively transported by the sodium, calcium, and proton pumps.
The specific aims are to identify amino acids involved in divalent cation binding, substrate binding, and monovalent cation binding and transport by the sodium pump. The first specific aim is to test a proposal for the magnesium binding site of sodium pump and the mechanism of phosphoryl group transfer made in the application. The topology of the magnesium site and the identity of the amino acids that coordinate magnesium are predicted by the model. The predictions will be tested by changing amino acids with side chains that interact with magnesium in the model and evaluating the effects of the mutations on a partial reaction (""""""""back door"""""""" phosphorylation) in which the phosphoryl group is transferred from the enzyme to water by measuring oxygen exchange between inorganic phosphate and water. The second specific aim is to change amino acids suspected of binding substrate by site-directed mutagenesis and study the effects of the mutations on back door oxygen exchange, as well as the overall catalytic cycle, to learn the specific reaction steps affected. The third specific aim is to test the hypothesis that the cytosolic domain in one conformation of the enzyme initially recognizes sodium and potassium, which can subsequently can be occulded by amino acids in transmembrane domains. The hypothesis will be tested by studying the effects of site- directed mutations on the rate of back door oxygen exchange, which depends on an equilibrium between sodium and potassium conformations of the enzyme. The conformational change occurs in two steps, ion binding and occlusion, for which intrinsic ion dissociation constants and the equilibrium constant between unoccluded and occluded conformations will be estimated.
