Cardiac steroids inhibit Na,K-ATPase and the related non-gastric H,K-ATPase, while they do not interact with gastric H,K-ATPase. Introducing an arginine, the residue present in the gastric H,K-ATPase, in the second extracellular loop at the corresponding position 334 in the human non-gastric H,K-ATPase (D334R mutation) rendered it completely resistant to 2 mM ouabain. The corresponding mutation (E319R) in alpha 1 Na,K-ATPase produced a similar to 2-fold increase of the ouabain IC50 in the ouabain-resistant rat alpha 1 Na,K-ATPase and a large decrease of the ouabain affinity of human alpha 1 Na,K-ATPase, on the other hand this mutation had no effect on the affinity for the aglycone ouabagenin. These results provide a strong support for the orientation of ouabain in its biding site with its sugar moiety interacting directly with the second extracellular loop. (c) 2007 Elsevier Inc. All rights reserved.