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 I-50'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. Two residues, glutamic acids 955 and 956 of the ouabain resistant alpha(1) isoform, were substituted with glutamine and aspartic acid. The cDNAs coding for these substitutions were transfected into ouabain-sensitive HeLa cells. Resistant cells were obtained when enzymes with these substitutions were expressed, suggesting that E955 and E956 are not required for ion transport. Cation dependence of ATPase activity of the altered enzymes demonstrated only mild changes in kinetic constants, supporting the notion that these two amino acids are not major determinants of cation binding.