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

Ouabain is a highly polar acid 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 similar to3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak (similar to7%) response, and the effects reversed similar to 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 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 H-3-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.