LIGAND-BINDING SITES OF NA,K-ATPASE

Our studies have concentrated on two aspects of the Na,K-ATPase, the f irst relates to the identification of amino acids involved in binding Na+ and K+ during the catalytic cycle and the second involves defining how cardiac glycosides inhibit the enzyme. To date, three amino acids , Ser775, Asp804 and Asp808, all located in transmembrane regions five and six, have been shown to play a major role in K+ binding. These fi ndings are based on site directed mutagenesis and expression studies. In order to understand how cardiac glycosides interact with the Na,K-A TPase, studies again involving mutagenesis coupled with expression hav e been used. More specifically, amino acid residues have been substitu ted in an ouabain sensitive a subunit using random mutagenesis, and th e ability of the resulting enzyme to confer resistance to ouabain sens itive cells was determined. Interestingly, the amino acids of the a su bunit which alter ouabain sensitivity cluster in two major regions, on e comprised of the first and second transmembrane spanning domains and the extracellular loop joining them, and the second formed by the ext racellular halves of transmembrane regions four, five, six and seven. As noted above, transmembrane regions five and six also contain the th ree amino acid residues Ser775, Asp804 and Asp808 which play a key rol e in cation transport, possibly binding K+. Thus, it is reasonable to propose that cardiac glycosides bind to two sites, the N- terminal reg ion and the central region which contains the cation binding sites. Ca rdiac glycoside binding to the center region may lock the cation trans port region into a configuration such that the enzyme cannot go throug h the conformational change required for ion transport.