Ouabain Binding Site in a Functioning Na  /K  ATPase * □ S Received for publication, June 2, 2011, and in revised form, September 5, 2011 Published, JBC Papers in Press, September 12, 2011, DOI 10.1074/jbc.M111.267682 Walter Sandtner ‡ , Bernhard Egwolf § , Fatemeh Khalili-Araghi ¶ , Jorge E. Sa ´ nchez-Rodríguez ¶1 , Benoit Roux ¶2 , Francisco Bezanilla ¶3 , and Miguel Holmgren 4 From the ‡ Department of Pharmacology, Medical University of Vienna, Waehringer Strasse 13A, 1090 Vienna, Austria, the § Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, 37077 Go ¨ttingen, Germany, the ¶ Department of Biochemistry and Molecular Biology, The University of Chicago Gordon Center for Integrative Science, Chicago, Illinois 60637, and the  Molecular Neurophysiology Section, Porter Neuroscience Research Center, NINDS, National Institutes of Health, Bethesda, Maryland 20892 Background: Ouabain binds at the permeation pathway of the Na + /K + ATPase. Results: We have identified two binding sites for ouabain along the ion conductive pathway of the Na + /K + pump that are mutually exclusive and differ in their affinities by about an order of magnitude. Conclusion: Ouabain reaches its high affinity binding site at the inner end of the permeation pathway by a sequential mechanism. Significance: This work unifies all available functional and structural data on the interactions of ouabain with the Na + /K + pump. The Na  /K  ATPase is an almost ubiquitous integral mem- brane protein within the animal kingdom. It is also the selec- tive target for cardiotonic derivatives, widely prescribed inhibitors for patients with heart failure. Functional studies revealed that ouabain-sensitive residues distributed widely throughout the primary sequence of the protein. Recently, structural work has brought some consensus to the functional observations. Here, we use a spectroscopic approach to esti- mate distances between a fluorescent ouabain and a lantha- nide binding tag (LBT), which was introduced at five different positions in the Na  /K  ATPase sequence. These five nor- mally functional LBT-Na  /K  ATPase constructs were expressed in the cell membrane of Xenopus laevis oocytes, operating under physiological internal and external ion con- ditions. The spectroscopic data suggest two mutually exclu- sive distances between the LBT and the fluorescent ouabain. From the estimated distances and using homology models of the LBT-Na  /K  ATPase constructs, approximate ouabain positions could be determined. Our results suggest that oua- bain binds at two sites along the ion permeation pathway of the Na  /K  ATPase. The external site (low apparent affinity) occupies the same region as previous structural findings. The high apparent affinity site is, however, slightly deeper toward the intracellular end of the protein. Interestingly, in both cases the lactone ring faces outward. We propose a sequential ouabain binding mechanism that is consistent with all func- tional and structural studies. Throughout their lives, animal cells maintain an imbalance of the concentrations of Na + and K + between their internal and external environments. These ionic gradients are used in cellu- lar processes that are essential for cell survival. On the one hand, the K + gradient maintains the negative resting potential. On the other hand, the Na + gradient is used by most cells to transport nutrients and metabolic substrates inside the cell, to extrude deleterious excess of intracellular ions, like Ca 2+ , and in excitable cells it allows the generation of action potentials. More than 40% of the energy produced in an animal cell is consumed by the Na + /K + ATPase to maintain the Na + and K + gradients. The Na + /K + ATPase is an integral membrane protein formed mainly by two subunits,  and . The larger (1,000 amino acids)  subunit spans 10 times across the cell mem- brane and contains all of the necessary components for ion transport: the ion permeation pathway, the phosphorylation site, and the ATP binding domain (1, 2). The smaller  subunit (300 amino acids) spans once through the membrane to form a large extracellular structure that sits on top of the external surface of the  subunit. The  subunit is essential for traffick- ing and K + transport (3, 4). Mutations within the Na + /K + ATPase have been implicated with human diseases like rapid-onset dystonia parkinsonism (5) and familial hemiplegic migraine (6 – 8). Clinically, however, the most important role of the Na + /K + ATPase is being the target of cardiotonic steroids in heart failure patients, a practice in use for more than two centuries. It is for this reason that a vast body of work has been devoted to identify the binding site of these compounds (9 –20), including the lately solved Na + /K + ATPase crystal structures with a molecule of ouabain bound (21, 22). In these structures the Na + /K + ATPase was * This work was supported, in whole or in part, by National Institutes of Health Grants R01-GM062342, R01-GM030376, and U54-GM087519. This work was also supported by the Intramural Research Program NINDS, National Institutes of Health. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S3. 1 Recipient of a postdoctoral fellowship from Consejo Nacional de Ciencia y Tecnología (Mexico). 2 To whom correspondence may be addressed: 929 E. 57 St., Chicago, IL 60637. Fax: 773-702-1330; E-mail: roux@uchicago.edu. 3 To whom correspondence may be addressed: 929 E. 57 St., Chicago, IL 60637. Fax: 773-702-1330; E-mail: fbezanilla@uchicago.edu. 4 To whom correspondence may be addressed: 35 Convent Dr., B35 Rm. 3B1016, Bethesda, MD 20892-3701. Fax: 301-496-4668; E-mail: holmgren@ ninds.nih.gov. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 44, pp. 38177–38183, November 4, 2011 Printed in the U.S.A. NOVEMBER 4, 2011 • VOLUME 286 • NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 38177 at University of Chicago Library, on November 22, 2011 www.jbc.org Downloaded from http://www.jbc.org/content/suppl/2011/09/12/M111.267682.DC1.html Supplemental Material can be found at: