Functional properties of an H,K-ATPase/Na,K-ATPase chimera.
Cultured pig kidney epithelial cells were transfected with a chimeric P-type ATPase catalytic subunit composed of the NH2-terminal half of the rat gastric H,K-ATPase and the COOH-terminal half of the rat Na,K-ATPase (alpha 1 isoform). Low concentrations of ouabain (< or = 0.2 mM) were used to inhibit completely the endogenous pig Na,K-ATPase and high concentrations (5 nM) to test the sensitivity of the chimeric rodent pump. In the presence of a low concentration of ouabain, a small but significant inhibition of residual Rb+(K+) influx by 5 mM ouabain was observed in only the transfected cells. Conditions were found in which a similar component of Rb+ influx was inhibited by the gastric H,K-ATPase inhibitor SCH28080, consistent with SCH28080 binding to the extracellular H1-H2 loop of this enzyme. These experiments demonstrate that this chimera behaves as a functional ion pump and indicate that the protein domains involved in cardiac glycoside binding are not confined to the amino-terminal half of the Na,K-ATPase.
Blostein
R
R
Department of Medicine, McGill University, Montreal, Quebec, Canada.
Zhang
R
R
Gottardi
C J
CJ
Caplan
M J
MJ
D000818:Animals; D000897:Anti-Ulcer Agents / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007093:Imidazoles / Q000494:pharmacology; D007668:Kidney / Q000378:metabolism; D007700:Kinetics; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D017433:Protein Structure, Secondary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013270:Stomach / Q000201:enzymology*; D013552:Swine; D014162:Transfection
D000897:Anti-Ulcer Agents; D002712:Chlorides; D007093:Imidazoles; D011993:Recombinant Fusion Proteins; C035235:Sch 28080; D010042:Ouabain; D012964:Sodium; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium; C032710:rubidium chloride; D011188:Potassium