Biological Implication of Conformational Flexibility in Ouabain:  Observations with Two Ouabain Phosphate Isomers

• Published in: Biochemistry (Easton) Vol. 40; no. 19; pp. 5835 - 5844
• Main Authors: Kawamura, Akira, Abrell, Leif M, Maggiali, Federica, Berova, Nina, Nakanishi, Koji, Labutti, Jason, Magil, Sheila, Haupert, Garner T, Hamlyn, John M
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 15.05.2001
• Subjects: Animals; Binding, Competitive; Biosensing Techniques; Carbohydrate Conformation; Carbohydrate Sequence; Cell Line; Dogs; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Isomerism; Kidney - enzymology; Kinetics; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Ouabain - analogs & derivatives; Ouabain - chemistry; Ouabain - metabolism; Phosphates - chemistry; Phosphates - metabolism; Sodium-Potassium-Exchanging ATPase - antagonists & inhibitors; Sodium-Potassium-Exchanging ATPase - metabolism; Tritium
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0101751

Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10-5 M reduced the rate of extracellular acidification by 15−20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was ∼3 times faster than ouabain. The 1,11,19-phosphate at 10-4 M elicited a weak (∼7%) response, and the effects reversed ∼44-fold faster than ouabain. Studies with purified Na+,K+-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC50 = 1.1 and 5.2 × 10-7 M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na+,K+-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.