Structure-function studies of the Na,K-ATPase

Na,K-ATPase catalyzes the movement of sodium and potassium ions across the cell membrane utilizing ATP as an energy source. This enzyme is present in almost all tissues of higher organisms but is most abundant in the kidney where it is responsible for reabsorbing sodium ions from the glomerular filtrate. The enzyme is composed of two subunits and serves as the receptor for cardiac glycosides. Utilizing an expression/selection system it has been possible to identify several amino acid residues that affect sensitivity to the cardiac glycoside, ouabain. Those identified to date are located in the first transmembrane region and first extracellular region. The fact that amino acid residues within a transmembrane region affect ouabain sensitivity suggests that the drug is partially internalized in the lipid bilayer. In an effort to determine whether any of the amino acid residues which affect ouabain sensitivity interact with the sugar part of cardiac glycosides, ouabain and ouabagenin were tested in terms of their ability to inhibit enzyme containing substitutions at these positions. The two compounds differ in that ouabagenin lacks a sugar moiety. Interestingly, the ratio of I50's for the substituted enzymes remains the same as the wild type, which suggests that the amino acids identified as determinants of ouabain sensitivity to date are not likely to interact with the sugar. Another set of studies focused on cation binding. It has been proposed that cation transport involves negatively charged residues in one or more transmembrane regions.(ABSTRACT TRUNCATED AT 250 WORDS)