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Keywords:

  • Na+/K+-exchanging ATPase;
  • cardiac glycoside;
  • ouabain;
  • random mutagenesis;
  • drug interaction

Abstract

  1. Top of page
  2. Abstract
  3. References

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the α1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111[RIGHTWARDS ARROW]Arg, Asp121[RIGHTWARDS ARROW]Gly, Asp121[RIGHTWARDS ARROW]Glu, Asn122[RIGHTWARDS ARROW]Asp, and Thr797[RIGHTWARDS ARROW]a were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330[RIGHTWARDS ARROW]Gln, Ala331[RIGHTWARDS ARROW]Gly, Thr338[RIGHTWARDS ARROW]a, and Thr338[RIGHTWARDS ARROW]Asn in the H4 transmembrane domain and Phe982[RIGHTWARDS ARROW]Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1–-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase α1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.

Abbreviation.
IC50

concentration of cardiac glycoside that produces 50% inhibition of activity

Enzyme.
 

Na+/K+-exchanging ATPase (EC 3.6.1.37)

References

  1. Top of page
  2. Abstract
  3. References
  • Argüello, J. M. & Kaplan, J. H. (1991) Evidence for essential carboxyls in the cation-binding domain of the Na, K-ATPase, J. Biol. Chem. 266, 1462714635.
  • Argüello, J. M. & Kaplan, J. H. (1994) Glutamate 779, an intramembrane carboxyl, is essential for monovalent cation binding by the Na, K-ATPase, J. Biol. Chem. 269, 68926899.
  • Argüello, J. M. & Lingrel, J. B. (1995) Substitution of serine 775 in the α subunit of the Na, K-ATPase selectivity disrupts K+ high affinity activation without affecting Na+ interaction, J. Biol. Chem. 270, 2276422771.
  • Argüello, J. M., Peluffo, R. D., Feng, J., Lingrel, J. B. & Berlin, J. (1996) Substitution of glutamic 779 with alanine in the Na, K-ATPase alpha subunit removes voltage dependence of ion transport, J. Biol. Chem. 271, 2461024616.
  • Askari, A., Kakar, S. & Huang, W. (1988) Ligand binding sites of the ouabain-complexed Na, K-ATPase, J. Biol. Chem. 263, 235242.
  • Askew, G. R. & Lingrel, J. B. (1994) Identification of an amino acid substitution in human α1 Na, K-ATPase which confers differentially reduced affinity for two related cardiac glycosides, J. Biol. Chem. 269, 2412024126.
  • Arystarkhova, E., Gasparian, M., Modyanov, N. M. & Sweader, K. J. (1992) Na, K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding, J. Biol. Chem. 267, 1369413701.
  • Blostein, R., Xhang, R., Gottardi, C. J. & Caplan, M. J. (1993) Functional properties of an H, K-4TPase/Na, K-ATPase chimera, J. Biol. Chem. 268, 1065410658.
  • Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72, 248254.
  • Burns, E. L. & Price, E. M. (1993) Random mutagenesis of the sheep Na, K-ATPase alpha-1 subunit generates a novel T797N mutation that results in a ouabain-resistant enzyme, J. Biol. Chem. 268, 2563225635.
  • Burns, E. L., Nicholas, R. A. & Price, E. M. (1996) Random mutagensis of the sheep Na, K-ATPase alpha 1 subunit generating the ouabain-resistant mutant L793P, J. Biol. Chem. 271, 1587915883.
  • Cadwell, R. C. & Joyce, G. F. (1992) Randomization of genes by PCR mutagenesis, PCR Methods Appl. 2, 2833.
  • Canessa, C. M., Horisberger, J.-D., Louvard, D. & Rossier, B. C. (1992) Mutation of a cysteine in the first transmembrane segment of Na, K-ATPase alpha subunit confers ouabain resistance, EMBO J. 11, 16811687.
  • Canessa, C. M., Horishberger, J.-D. & Rosier, B. C. (1993) Mutation of a tyrosine in the H3-H4 ectodomain of Na, K-ATPase a subunit confers ouabain resistance, J. Biol. Chem. 268, 1772217726.
  • Canfield, V., Emanuel, J. R., Speckofsky, N., Levenson, R. & Margolskee, T. E. (1990) Ouabain-resistant mutants of the rat Na, K-AT-Pase alpha 2 isoform identified by using an episomal expression vector, Mol. Cell. Biol. 10, 13671372.
  • Chantley, L. G., Cunha, M. J. & Zhour, X.-M. (1994) Ouabain-resistant OR6 cells express the murine α1 subunit of Na, K-ATPase with T797I substitution, J. Biol. Chem. 269, 1535815361.
  • Erdmann, E. & Schoner, W. (1973) Ouabain-receptor interactions in (Na, K)-ATPase preparations 3. On the stability ofthe ouabain receptor against physical treatment, hydrolases, and SH reagents, Biochim. Biophys. Acta. 330, 302315.
  • Feller, W. (1970) An introduction to probability theory and its application, 3rd edn, Wiley Publishing Co., NY .
  • Feng, J. & Lingrel, J. B. (1994) Analysis of amino acid residues in the H5–H6 transmembrane and extracellular domain of the Na, K-ATPase subunit identified T797 as a determinant of ouabain sensitivity, Biochemistry 33, 42184224.
  • Forbush, B. (1983) Cardiotonic steroid binding to Na, K-ATPase, in Current topics on membranes and transport (Hoffman, J. F. & Forbush, B.III, eds) vol. 19, pp. 167201, Academic Press, New York .
  • Hansen, O. (1984) Interaction of cardiac glycosides with Na, K-activated ATPase. A biochemical link to digitalis-induced inotropy, Pharmacol. Rev. 36, 143163.
  • Ishii, T. & Takeyasu, R. (1993) The amino-terminal 200 amino acids of the plasma Na, K-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase, Proc. Natl Acad. Sci. USA 90, 88818885.
  • Jewell, E. A. & Lingrel, J. B. (1991) Comparison of the substrate dependence properties of the rat Na, K-ATPase α1, α2, and α3 expressed in HeLa cells, J. Biol Chem. 266, 1692516930.
  • Karlish, S. J. D., Goldschleger, R. & Jorgensen, P. L. (1993) Location of Asn 831 of the α chain of Na, K-ATPase at the cytoplasmic surface. Implication for topological models, J. Biol. Chem. 268, 34713478.
  • Kirley, T. L., Lane, L. K. & Wallick, E. T. (1986) Identification of an essential sulfhydryl groups in the ouabain site of (Na, K)-ATPase, J. Biol. Chem. 261, 45254528.
  • Kirley, T. L. & Peng, M. (1991) Identification of cysteine residues in lamb kidney (Na, K)-ATPase essential for ouabain binding, J. Biol. Chem. 266, 1995319957.
  • Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenses without phenoypic selection, Proc. Natl Acad. Sci. USA 82, 488492.
  • Kuntzweiller, T. A., Argüello, J. M. & Lingrel, J. B. (1996) Asp804 and Asp808 in the transmembrane domain of the Na, K-ATPase a subunit are cation coordinating residues, J. Biol. Chem. 271, 2968229687.
  • Lanzetta, P. A., Alvarex, L. J., Reinach, P. S. & Candia, O. A. (1979) An improved assay for nanomole amounts of inorganic phosphate, Anal. Biochem. 100, 9597.
  • Lingrel, J. B., Argüello, J. M., Van Huysse, J. & Kuntzweiler, T. A. (1997) Cation and cardiac glycoside binding sites of the Na, K-AT-Pase, in Na, K-ATPase and related transport ATPases: structure, mechanism and regulation (Beauge, L. A., Garrahan, P. J. & Gadsby, D. C., eds) in the press, New York Academy of Sciences, New York .
  • Lingrel, J. B., Orlowski, J., Price, E. M. & Pathak, B. G. (1991) Regulation of the α-subunit genes of the Na, K-ATPase and determinants of cardiac glycoside sensitivity, in The sodium pump: structure, mechanism and regulation (Kaplan, J. H. & DeWeer, P., eds) pp. 116, The Rockefeller University Press.
  • Lingrel, J. B. & Kuntzweiler, T. A. (1994) Na, K-ATPase mini review, J. Biol. Chem. 269, 1965919662.
  • Lutsenko, S. & Kaplan, J. H. (1994) Molecular events in close proximity to the membrane associated with the binding of ligands to the Na, K-ATPase, J. Biol. Chem. 269, 45554564.
  • Lutsenko, S., Anderko, R. & Kaplan, J. H. (1995) Membrane disposition of the M5–M6 hairpin of Na, K-ATPase alpha subunit is ligand dependent, Proc. Natl Acad. Sci. USA 92, 79367940.
  • Malik, B., Jamieson, G. A. & Ball, W. Jr (1993) Identification of the amino acids comprising of surface-exposed epitope within the nucleotide-binding domain of the Na, K-ATPase using a random pepetide library, Protein Sci. 2, 21032111.
  • Palasis, M., Kuntzweiler, T. A., Argüello, J. M. & Lingrel, J. B. (1996) Ouabain interactions with the H5-H6 hairpin of Na, K-ATPase reveals a possible inhibition mechanism via the cation binding domain, J. Biol. Chem. 271, 1417614182.
  • Price, E. & Lingrel, J. B. (1988) Structure-function relationships in the Na, K-ATPase α subunit: sited directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain resistant enzyme, Biochemistry 27, 84008408.
  • Price, E. M., Rice, D. A. & Lingrel, J. B (1989) Site directed mutagensis of a conserved extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na, K-ATPase, J. Biol. Chem. 264, 2190221906.
  • Price, E. M., Rice, D. A. & Lingrel, J. B. (1990) Structure-function studies of Na, K-ATPase, J. Biol. Chem. 265, 66386642.
  • Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular cloning: a laboratory manual, 2nd edn, pp. 18601874, Cold Spring Harbor Laboratory Press, Plainview , NY .
  • Schultheis, P. J. & Lingrel, J. B. (1993) Substitution of transmembrane residues with hydrogen-bonding potential in the a subunit of Na, K-ATPase reveals alterations in ouabain binding, Biochemistry 32, 544550.
  • Schultheis, P. J., Wallick, E. T. & Lingrel, J. B. (1993) Kinetic analysis of ouabain binding to native and mutated forms of Na, K-ATPase and identification of a new region involved in cardiac glycoside interaction, J. Biol. Chem. 268, 2268622694.
  • Thomas, R., Gray, P. & Andrews, J. (1990) Digitalis: its mode of action, receptor, and structure-activity relationships, Adv. Drug Res. 19, 311562.
  • Yoda, A. & Yoda, S. (1982) Interactions between ouabain and the phosphorylated intermediate of Na, K-ATPase, Mol. Pharmacol. 22, 700705.