Significance: Na ⁺ ,K ⁺ -ATPase is the ion pump responsible for maintenance of the electrochemical gradients of Na ⁺ and K ⁺ across the membrane of animal cells. Cardiotonic steroids constitute a broad class of specific Na ⁺ ,K ⁺ -ATPase inhibitors, including drugs of clinical importance with multiple physiological effects. The existence of several endogenous cardiotonic steroids suggests their involvement in health and disease mediated by various signaling pathways, but the structure–activity relationships are not yet understood. Using X-ray crystallography and analysis of binding kinetics, we characterize Na ⁺ ,K ⁺ -ATPase complexes with a total of five cardiotonic steroids, showing variations in glycosylation, steroid core substituents, and structure of the lactone substituent. This insight is highly relevant for the understanding of physiological effects and future drug development based on cardiotonic steroids.

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 ⁺ ,K ⁺ -ATPase. Here we describe a crystal structure of the phosphorylated pig kidney Na ⁺ ,K ⁺ -ATPase in complex with the CTS representative ouabain, extending to 3.4 Å resolution. The structure provides key details on CTS binding, revealing an extensive hydrogen bonding network formed by the β-surface of the steroid core of ouabain and the side chains of αM1, αM2, and αM6. Furthermore, the structure reveals that cation transport site II is occupied by Mg ²⁺ , and crystallographic studies indicate that Rb ⁺ and Mn ²⁺ , but not Na ⁺ , bind to this site. Comparison with the low-affinity [K ₂ ]E2–MgF _x –ouabain structure [Ogawa et al. (2009) Proc Natl Acad Sci USA 106(33):13742–13747) shows that the CTS binding pocket of [Mg]E2P allows deep ouabain binding with possible long-range interactions between its polarized five-membered lactone ring and the Mg ²⁺ . K ⁺ binding at the same site unwinds a turn of αM4, dragging residues Ile318–Val325 toward the cation site and thereby hindering deep ouabain binding. Thus, the structural data establish a basis for the interpretation of the biochemical evidence pointing at direct K ⁺ –Mg ²⁺ competition and explain the well-known antagonistic effect of K ⁺ on CTS binding.

Pumping Out Sodium: Mammalian cells contain relatively high concentrations of potassium but low concentrations of sodium. This balance is maintained by an ion pump, the Na ⁺ , K ⁺ –adenosine triphosphatase, in an adenosine triphosphate–driven transport cycle that results in the export of three sodium ions and the import of two potassium ions. Structures of potassium-bound conformations of the pump have been determined. Now, Nyblom et al. (p. 123 , published online 19 September) report on the high-resolution crystal structure of a Na ⁺ -bound conformation, which reveals conformational changes associated with Na ⁺ binding.