Interaction between Cardiotonic Steroids and Na,K-ATPase. Effects of pH and Ouabain-Induced Changes in Enzyme Conformation

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. It maintains the transmembrane gradients of Na(+) and K(+) across the cell membrane essential for cell homeostasis. The Na,K-ATPase is specifically inhibited by cardiotonic steroids like ouabain, which bind to the extracellular side of the enzyme and is of significant therapeutic value in the treatment of congestive heart failure. In order to further characterize the binding of cardiotonic steroids to shark Na,K-ATPase, we compared the strength and rate of inhibition at varying pH of two cardiac glycosides with either an unsaturated (ouabain) or saturated (dihydroouabain) lactone ring and three aglycons with either a 5-membered (ouabagenin and digitoxigenin) or a 6-membered (bufalin) lactone. Inhibition by ouabain and dihydroouabain, and especially the aglycon ouabagenin, was found to be strongly dependent on pH with an increase in IC(50) by factors of approximately 6, approximately 20, and approximately 66, respectively, when pH increased from 6.5 to 8.5. The finding that ouabagenin was the most pH-sensitive inhibitor indicates that the steroid hydroxyl side chains are pivotal for this pH effect, whereas the lactone ring saturation was less important. The sugar moiety is important in compensating for the pH effect. In contrast, the IC(50) of the two genins bufalin and digitoxigenin increased by a factor of only approximately 2 when pH increased from 6.5 to 8.5, indicating that the pH effect does not relay on whether the lactone is 5- or 6-membered. The rate of inhibition was retarded much more significantly by increasing pH for the glycosides than for the aglycons. Finally, we demonstrate a change in enzyme subconformations following binding of cardiotonic steroids to Na,K-ATPase phosphoenzymes using fluoride analogues of phosphoenzyme intermediates. The results are discussed with reference to the recent high-resolution crystal structures of shark Na,K-ATPase in the unbound and ouabain-bound conformation.