Phe783, Thr797, and Asp804 in Transmembrane Hairpin M5-M6 of Na+,K+-ATPase Play a Key Role in Ouabain Binding

Abstract

Ouabain is a glycoside that binds to and inhibits the action of Na ؉ ,K ؉ -ATPase. Little is known, however, about the specific requirements of the protein surface for glycoside binding. Using chimeras of gastric H ؉ ,K ؉ -ATPase and Na ؉ ,K ؉ -ATPase, we demonstrated previously that the combined presence of transmembrane hairpins M3-M4 and M5-M6 of Na ؉ ,K ؉ -ATPase in a backbone of H ؉ ,K ؉ -ATPase (HN34/56) is both required and sufficient for high affinity ouabain binding. Since replacement of transmembrane hairpin M3-M4 by the N terminus up to transmembrane segment 3 (HNN3/56) resulted in a low affinity ouabain binding, hairpin M5-M6 seems to be essential for ouabain binding. To assess which residues of M5-M6 are required for ouabain action, we divided this transmembrane hairpin in seven parts and individually replaced these parts by the corresponding sequences of H ؉ ,K ؉ -ATPase in chimera HN34/56. Three of these chimeras failed to bind ouabain following expression in Xenopus laevis oocytes. Altogether, these three chimeras contained 7 amino acids that were specific for Na ؉ ,K ؉ -ATPase. Individual replacement of these 7 amino acids by the corresponding amino acids in H ؉ ,K ؉ -ATPase revealed a dramatic loss of ouabain binding for F783Y, T797C, and D804E. As a proof of principle, the Na ؉ ,K ؉ -ATPase equivalents of these 3 amino acids were introduced in different combinations in chimera HN34. The presence of all 3 amino acids appeared to be required for ouabain action. Docking of ouabain onto a three-dimensional-model of Na ؉ ,K ؉ -ATPase suggests that Asp 804 , in contrast to Phe 783 and Thr 797 , does not actually form part of the ouabain-binding pocket. Most likely, the presence of this amino acid is required for adopting of the proper conformation for ouabain binding.

Ouabain affinity determining residues lie close to the Na/K pump ion pathway

“…And mutating that Thr to Cys renders the Na͞K pump susceptible to inhibition by omeprazole (31), while making it less sensitive to inhibition by ouabain (31,36). This correlation, together with similarities between omeprazole inhibition of H͞K pumps and cardiotonic steroid inhibition of Na͞K pumps, supports the idea that cardiotonic steroids dock in the space above TM6 shown in Fig.…”

Reconstruction of the Complete Ouabain-binding Pocket of Na,K-ATPase in Gastric H,K-ATPase by Substitution of Only Seven Amino Acids

“…In a previous study, we demonstrated that replacement of the transmembrane hairpins M3-M4 and M5-M6 in H,KATPase by those of Na,K-ATPase results in the formation of a high affinity ouabain-binding site (10). In a follow-up study we showed that a chimera, in which only the M3-M4 hairpin and three amino acids of M5-M6 (Phe 783 , Thr 797 , and Asp 804 ) originate from Na,K-ATPase, binds ouabain with high affinity (11). However, the M3-M4 hairpin in the latter chimera still contains 27 amino acids that are unique for Na,KATPase.…”

“…Binding, and ATP Phosphorylation-Oocytes were injected with cRNAs, and after 3 days the membranes were isolated as described previously (11). For immunoblotting, the ␣-subunits of H,K-ATPase and the chimeras were detected with the polyclonal antibody HKB (15).…”

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase

“…The complexes were labelled with a score that approximates binding energy. Because of the huge size of the ATPase model, placement of the ligand was restricted to the previously postulated binding pocket on the extracellular side of the membrane [12]. Besides an overall pocket, a sub-pocket was defined to include residues known from mutation experiments to be important for ligand binding.…”

“…The four transmembrane segments originating from Na,K-ATPase still contained 48 amino acids that were specific for this enzyme. Later, we could reduce this number to seven and still kept a high affinity for ouabain [12,13]. In the latter studies, we used the oocyte expression system and restricted ourselves to the measurement of ouabain binding.…”