Biased Effect of Cardiotonic Steroids on Na/K-ATPase–Mediated Signal Transduction

Recent studies have revealed that Na/K-ATPase (NKA) can transmit signals through ion-pumping-independent activation of pathways relayed by distinct intracellular protein/lipid kinases, and endocytosis challenges the traditional definition that cardiotonic steroids (CTS) are NKA inhibitors. Although additional effects of CTS have long been suspected, revealing its agonist impact through the NKA receptor could be a novel mechanism in understanding the basic biology of NKA. In this study, we tested whether different structural CTS could trigger different sets of NKA/effector interactions, resulting in biased signaling responses without compromising ion-pumping capacity. Using purified NKA, we found that ouabain, digitoxigenin, and somalin cause comparable levels of NKA inhibition. However, although endogenous ouabain stimulates both protein kinases and NKA endocytosis, digitoxigenin and somalin bias to protein kinases and endocytosis, respectively, in LLC-PK1 cells. The positive inotropic effects of CTS are traditionally regarded as NKA inhibitors. However, CTS-induced signaling occurs at concentrations at least one order of magnitude lower than that of inotropy, which eliminates their well known toxic actions on the heart. The current study adds a novel mechanism that CTS could exert its biased signaling properties through the NKA signal transducer. SIGNIFICANCE STATEMENT: Although it is now well accepted that NKA has an ion-pumping-independent signaling function, it is still debated whether direct and conformation-dependent NKA/effector interaction is a key to this function. Therefore, this investigation is significant in advancing our understanding of the basic biology of NKA-mediated signal transduction and gaining molecular insight into the structural elements that are important for cardiotonic steroid's biased action.