Amino Acid Residues Involved in Ouabain Sensitivity and Cation Binding

  • Jerry B. Lingrel
  • James Van Huysse
  • William O’Brien
  • Elizabeth Jewell-Motz
  • Patrick Schultheis
Conference paper
Part of the NATO ASI Series book series (NATO ASI, volume 89)

Abstract

The Na, K-ATPase, which is found in the cells of all higher eukaryotes, utilizes ATP to transport Na+ and K+ across the cell membrane. For every three sodium ions transported out of the cell two potassium ions are transported in. The enzyme is composed of two subunits, a larger a subunit, which is thought to contain most of the catalytic sites, and a smaller a subunit which is required for the proper processing and maturation of the enzyme. Three isoforms exist for the a subunit (α1, α2 and α3) and two isoforms exist for the β subunit (β1 and β2) in mammalian cells (Lingrel et al., 1990; Sweadner, 1989; Takeyasu et al, 1989). An additional isoform, β3, exists in Xenopous (Good et al., 1990). The a1 isoform is found in all cells while α2 is the primary isoform found in skeletal muscle, but is also present in the heart and nervous system. Expression of the α3 isoform is limited to the heart and nervous system (Orlowski and Lingrel, 1988). The cDNAs and genes corresponding to these isoforms have been isolated and the mechanisms responsible for their differential expression are being investigated (Lingrel et al., 1990). The Na,K-ATPase is a member of the P-type family of ATPases, which also include the Ca-ATPases and the H,K-ATPases. These transport proteins are similar in structure and have in common an aspart/l phosphate intermediate during their catalytic cycle. Site-directed mutagenesis and expression (MacLennan, 1990; Clarke et al., 1990; Vilsen and Andersen, 1992; Andersen and Vilsen, 1992) studies have been carried out to define cation binding sites in the Ca-ATPase but, less information is available using this approach with the Na.K-ATPase. The Na,K- ATPase differs from the other P-type ATPases in that it is sensitive to cardiac glycosides, a class of drugs which are used in the treatment of congestive heart failure and certain arrhythmias. The sensitivity of the enzyme to these drugs provides a useful tool for investigating structure-function relationships. In this report we describe progress made toward identifying potential cation binding sites as well as amino acid residues that are involved in determining cardiac glycoside sensitivity.

Keywords

Amino Acid Residue HeLa Cell Cardiac Glycoside Cation Binding Glutamic Acid Residue 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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References

  1. Andersen, J.P. and Vilsen, B. (1992) Functional consequences of alterations to GIu309, Glu771, and Asp800 in the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 267: 19383–19387PubMedGoogle Scholar
  2. Canessa, C.M., Horisberger, J.-D., Louvard, D. and Rossier, B.C. (1992) Mutation of a cysteine in the first transmembrane segment of Na,K-ATPase a subunit confers ouabain resistance. Embo J. 11: 1681–1687PubMedPubMedCentralGoogle Scholar
  3. Goldshleger, R., Tal, D.M., Moorman, J., Stein, W.D. and Karlish, S.J.D. (1992) Chemical modification of Glu-953 of the α chain of Na+, K+-ATPase associated with inactivation of cation occlusion. Proc. Natl. Acad. Sci. USA 89: 6911–6915CrossRefPubMedPubMedCentralGoogle Scholar
  4. Good, P.J., Richter, K. and Dawid, I.B. (1990) A nervous system-specific isotype of the beta subunit of Na,K-ATPase expressed during early development of Xenopus laevis. Proc. Natl. Acad. Sci. USA 87: 9088–9092CrossRefPubMedPubMedCentralGoogle Scholar
  5. Karlish, S.J.D., Goldshleger,R. and Stein, W.D. (1990) A 19- kDA C-terminal tryptic fragment of the a chain of Na/K-ATPase is essential for occlusion and transport of cations. Proc. Natl. Acad. Sci. USA 87: 4566–4570Google Scholar
  6. Kent, R.B., Emanuel, J.R., Neriah, Y.B., Levenson, R. and Housman, D.E. (1987) Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+- ATPase α subunit. Science 237: 901–903CrossRefPubMedGoogle Scholar
  7. Lingrel, J.B, Orlowski, J., Shull, M.M., and Price, E.M. (1990) Molecular Genetics of Na, K-ATPase. In: Progress in Nucleic Acids Research and Molecular Biology, Academic Press, Inc., 38: 37–89Google Scholar
  8. MacLennan, D.H. Molecular tools to elucidate problems in excitraction coupling. (1990) Biophys. J. 58: 1355–1365CrossRefGoogle Scholar
  9. MacLennan, D.H., C.J. Brandl, B. Korczak and N.M. Green. (1985) Amino-acid sequence of a Ca2+, Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence. Nature 316: 696–700CrossRefPubMedGoogle Scholar
  10. O’Brien, W.J., Wallick, E.T. and Lingrel, J.B. (1993) Amino acid residues of the Na, K- ATPase involved in ouabain sensitivity do not bind the sugar moiety of cardiac glycosides. J. Biol. Chem. 268: 7707–7712PubMedGoogle Scholar
  11. Orlowski, J. and Lingrel, J.B. (1988) Tissue-specific and developmental regulation of rat Na,K-ATPase catalytic a isoform and β subunit mRNAs. J. Biol. Chem. 263:10436- 10442Google Scholar
  12. Ovhinnikov, Y.A. (1987) Probing the folding of membrane proteins. Trends Biochem. Sci. 12: 434–438Google Scholar
  13. Pedemonte, C.H. and Kaplan, J.H. (1990) Chemical modification as an approach to elucidation of sodium pump structure-function relations. Am. J. Physiol. 258: C1–C23PubMedGoogle Scholar
  14. Price, E.M. and Lingrel, J.B. (1888) Structure-Function Relationships in the Na, K- ATPase a Subunit: Site-Directed Mutagenesis of Glutamine-111 to Arginine and Asparagine-122 to Aspartic Acid Generates a Ouabain-Resistant Enzyme. Biochemistry 27: 8400 - 8408CrossRefGoogle Scholar
  15. Price, E.M., Rice, D.A. and Lingrel, J.B. (1989) Site- directed mutagenesis of a conserved extracellular aspartic acid residue affects the ouabain sensitivity of sheep Na,K-ATPase. J. Biol. Chem. 264: 21902–21906Google Scholar
  16. Price, E.M., Rice, D.A. and Lingrel, J.B. (1990) Structure- function studies of Na, K- ATPase. J. Biol. Chem. 265: 6638–6641PubMedGoogle Scholar
  17. Schultheis, P.J. and Lingrel, J.B. (1993) Substitution of transmembrane residues with hydrogen-bonding potential in the α subunit of Na, K-ATPase reveals alterations in ouabain sensitivity. Biochemistry, 32: 544–550CrossRefPubMedGoogle Scholar
  18. Schultheis, P.J., Wallick, E.T. and 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 interactions. J. Biol. Chem. in press.Google Scholar
  19. Sweadner, K.J. (1989) Isozymes of the Na, K-ATPase. Biochem. Biophys. Acta., 988: 185–220PubMedGoogle Scholar
  20. Takeyasu, K., Renard, K.J., and. Aormino, J., Wolityky, B.A., Barnstein, A, Tamkum, M.M., and Fanbrough, D.M. (1989) Differential subunit and isoform expression involved in regulation of sodium pump in skeletal muscle. In Current Topics in Membranes and Transport. Academic Press, Inc.,New York, 143–165Google Scholar
  21. Van Huysse, J.W., Jewell, E.A., and Lingrel J.B. (1993) Site Directed Mutagenesis of a Predicted Cation Binding Site of Na, K-ATPase. Biochemistry, 32: 819–826Google Scholar
  22. Vilsen, B. and Andersen, J.P. (1992) CrATP-induced Ca2+ occlusion in mutants of the Ca2+-ATPase of sarcoplasmic reticulum. J. Biol. Chem. 267: 25739–25743PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1994

Authors and Affiliations

  • Jerry B. Lingrel
    • 1
  • James Van Huysse
    • 1
  • William O’Brien
    • 1
  • Elizabeth Jewell-Motz
    • 1
  • Patrick Schultheis
    • 1
  1. 1.Department of Molecular Genetics, Biochemistry and MicrobiologyUniversity of Cincinnati College of MedicineCincinnatiUSA

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