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Updated: Aug 9, 2026

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Published on: May 29, 2011
Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers
A Kawamura1, L M Abrell, F Maggiali
1Department of Chemistry, Columbia University, New York, New York 10027, USA.
Abstract:
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 approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 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 (EC(50) = 1.1 and 5.2 x 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.
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