Functional properties of an H,K-ATPase/Na,K-ATPase chimera

• Published in: The Journal of biological chemistry 1993-05, Vol.268 (14), p.10654-10658
• Main Authors: Blostein, R., Zhang, R., Gottardi, C.J., Caplan, M.J.
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Anti-Ulcer Agents - pharmacology; Biological and medical sciences; Biological Transport - drug effects; Cells, Cultured; Chlorides - metabolism; Enzymes and enzyme inhibitors; Epithelium - drug effects; Epithelium - metabolism; Fundamental and applied biological sciences. Psychology; H(+)-K(+)-Exchanging ATPase - chemistry; H(+)-K(+)-Exchanging ATPase - genetics; H(+)-K(+)-Exchanging ATPase - metabolism; Hydrolases; Imidazoles - pharmacology; Kidney - metabolism; Kinetics; Ouabain - pharmacology; Potassium - metabolism; Protein Structure, Secondary; Rats; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Rubidium - metabolism; Sodium - metabolism; Sodium-Potassium-Exchanging ATPase - chemistry; Sodium-Potassium-Exchanging ATPase - genetics; Sodium-Potassium-Exchanging ATPase - metabolism; Stomach - enzymology; Swine; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)82247-5

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.