High-affinity ouabain binding by a chimeric gastric H+,K+-ATPase containing transmembrane hairpins M3-M4 and M5-M6 of the alpha 1-subunit of rat Na+,K+-ATPase.
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 alpha-subunit of H(+),K(+)-ATPase were replaced by their counterparts of the alpha(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, 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.
Koenderink
J B
JB
Department of Biochemistry, Institute of Cellular Signaling, University of Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands.
Hermsen
H P
HP
Swarts
H G
HG
Willems
P H
PH
De Pont
J J
JJ
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D010042:Ouabain / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013270:Stomach / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D010042:Ouabain; D017506:H(+)-K(+)-Exchanging ATPase