Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na + ,K + -ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five-or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na + ,K + -ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg 2+ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K + in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.Na/K-ATPase | phosphoenzyme | inhibitor | cardiac glycosides | structure C ardiotonic steroids (CTSs) induce diverse physiological effects on, for example, heart muscle and blood pressure regulation, but the underlying mechanisms remain unknown, despite a long history of therapeutic applications and model studies. It is widely recognized that they target Na + ,K + -ATPase, and a direct consequence of their binding is an inhibition of the enzyme. Their positive inotropic effect in cardiomyocytes has been related to coupling between Na + ,K + -ATPase and Na + /Ca 2+ -exchanger through the intracellular Na + concentration, whereas numerous other outcomes observed on the cellular level have led to hypotheses of the existence of signaling cascade mechanisms with Na + ,K + -ATPase acting as a receptor. The minimal functional unit of the enzyme is an αβ-complex, and because there exist four α-and three β-isoforms of the Na + ,K + -ATPase, the variations in the heterodimer composition and a vast number of CTSs differing in apparent isoform specificities (1) add to the complexity and multiplicity of reported physiological responses.The conserved structural core shared by all CTSs includes a cis-trans-cis ring-fused steroid core with two methyl substituents at steroid positions C10 β and C13 β , two hydroxyl groups (OH3 β and OH14 β ), and an unsaturated lactone ring at the C17 β position, among which the lactone and OH14 β are critical for binding to Na + ,K + -ATPase (2, 3). The type of lactone at the C...

The Na + ,K + -ATPase maintains electrochemical gradients for Na + and K + that are critical for animal cells. Cardiotonic steroids (CTSs), widely used in the clinic and recently assigned a role as endogenous regulators of intracellular processes, are highly specific inhibitors of the Na

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P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.