Open AccessHigh-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
Author(s) -
Jan B. Koenderink,
H.P.H. Hermsen,
H.G.P. Swarts,
Peter H.G.M. Willems,
J.J.H.H.M. de Pont
Publication year - 2000
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.200109597
Subject(s) - ouabain , atpase , dephosphorylation , chemistry , h(+) k(+) exchanging atpase , protein subunit , enzyme , stereochemistry , amino acid , transmembrane protein , transmembrane domain , biochemistry , phosphatase , sodium , receptor , organic chemistry , gene
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 α-subunit of H+ ,K+ -ATPase were replaced by their counterparts of the α1 -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, HN34/56, 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 HN34/56 chimera, the M1/M2 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 M1/M2 loop of chimera HN34/56 results in a decreased ouabain affinity. This indicates that although the M1/M2 loop affects the ouabain affinity, binding occurs when the M3/M4 and M5/M6 hairpins of Na+ ,K+ -ATPase are present.