Biological implication of conformational flexibility in ouabain: Observations with two ouabain phosphate isomers

Akira Kawamura, Leif M. Abrell, Federica Maggiali, Nina Berova, Koji Nakanishi, Jason Labutti, Sheila Magil, Garner T. Haupert, John M. Hamlyn

Research output: Contribution to journalArticlepeer-review

26 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)5835-5844
Number of pages10
Issue number19
StatePublished - May 15 2001

ASJC Scopus subject areas

  • Biochemistry


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