High-affinity ouabain binding by a chimeric gastric H ⁺ ,K ⁺ -ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the α ₁ -subunit of rat Na ⁺ ,K ⁺ -ATPase

Abstract

Na ؉ ,K ؉ -ATPase and gastric H ؉ ,K ؉ -ATPase are two related enzymes that are responsible for active cation transport. Na ؉ ,K ؉ -ATPase activity is inhibited specifically by ouabain, whereas H ؉ ,K ؉ -ATPase is insensitive to this drug. Because it is not known which parts of the catalytic subunit of Na ؉ ,K ؉ -ATPase are responsible for ouabain binding, we prepared chimeras in which small parts of the ␣-subunit of H ؉ ,K ؉ -ATPase were replaced by their counterparts of the ␣1-subunit of rat Na ؉ ,K ؉ -ATPase. A chimeric enzyme in which transmembrane segments 5 and 6 of H ؉ ,K ؉ -ATPase were replaced by those of Na ؉ ,K ؉ -ATPase could form a phosphorylated intermediate, but hardly showed a K ؉ -stimulated dephosphorylation reaction. When transmembrane segments 3 and 4 of Na ؉ ,K ؉ -ATPase were also included in this chimeric ATPase, K ؉ -stimulated dephosphorylation became apparent. This suggests that there is a direct interaction between the hairpins M3-M4 and M5-M6. Remarkably, this chimeric enzyme, HN34͞56, had obtained a high-affinity ouabain-binding site, whereas the rat Na ؉ ,K ؉ -ATPase, from which the hairpins originate, has a low affinity for ouabain. The low affinity of the rat Na ؉ ,K ؉ -ATPase previously had been attributed to the presence of two charged amino acids in the extracellular domain between M1 and M2. In the HN34͞56 chimera, the M1͞M2 loop, however, originates from H ؉ ,K ؉ -ATPase, which has two polar uncharged amino acids on this position. Placement of two charged amino acids in the M1͞M2 loop of chimera HN34͞56 results in a decreased ouabain affinity. This indicates that although the M1͞M2 loop affects the ouabain affinity, binding occurs when the M3͞M4 and M5͞M6 hairpins of Na ؉ ,K ؉ -ATPase are present.

Progesterone binding to the α1-subunit of the Na/K-ATPase on the cell surface: Insights from computational modeling

“…The crystallographic data may, however, predict an arrangement of the ten α1-subunit helices within the plane of the lipid bilayer that is common to both enzymes. In a third approach, chimeras of individual Na/KATPase loop regions were inserted into either the skeletal muscle sarcoplasmic reticulum/ endoplasmic reticulum Ca 2+ -ATPase (SERCA1) [39,40] or the gastric H/K-ATPase [41,42]. The results suggest that M1-M2, M3-M4 and M5-M6 extracellular loops (see Figure 2) are important for ouabain sensitivity.…”

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

“…The ATPase activity of chimera HN34/56 was 49% of that of the wild type H,K-ATPase at 10 M ATP, 3 mM KCl, and pH 6.0 (10). The ouabain binding level of Na,K-ATPase (QN) was 0.40 Ϯ 0.04 pmol/mg when 1 mM P i was present, whereas that of HN34/56 was 0.20 Ϯ 0.01 pmol/mg when 1 mM ATP was present (10). The ouabain binding level of H,K-ATPase was only 0.04 Ϯ 0.01 pmol/mg in the presence of 1 mM P i or ATP.…”

“…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).…”

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

“…The chimeras and mutants used in this study were: the rat Na,K-ATPase α 1 -subunit (wild type), the R111Q/ D122N mutant (QN) of this enzyme [10,11], the rat non-gastric H, K-ATPase α 2 -subunit [13] as well as its EGPLC mutant (D312E, S319G, A778P, I795L, F802C) [14]. Generation of the vectors containing the α 2 -subunit of rat non-gastric H,K-ATPase with the β 1 -subunit of rat Na,K-ATPase or the α 1 -subunit of rat Na,K-ATPase with the β 1 -subunit of sheep Na,K-ATPase that were suited for the baculovirus expression system, has been reported before [5,14].…”

“…In 2000, we surprisingly showed a high affinity for ouabain in a chimera that had, as basis, the gastric H,K-ATPase (α and β subunits) and in which the transmembrane segments M3/ M4 and M5/M6 of the α-subunit were replaced by those of Na,K-ATPase [11]. This chimera was expressed in insect cells using the baculovirus expression system.…”