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. 1987 Oct 1;105(4):1855–1865. doi: 10.1083/jcb.105.4.1855

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications

PMCID: PMC2114652  PMID: 2822726

Abstract

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

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Selected References

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  1. Adams G. A., Rose J. K. Structural requirements of a membrane-spanning domain for protein anchoring and cell surface transport. Cell. 1985 Jul;41(3):1007–1015. doi: 10.1016/s0092-8674(85)80081-7. [DOI] [PubMed] [Google Scholar]
  2. Baron R., Neff L., Roy C., Boisvert A., Caplan M. Evidence for a high and specific concentration of (Na+,K+)ATPase in the plasma membrane of the osteoclast. Cell. 1986 Jul 18;46(2):311–320. doi: 10.1016/0092-8674(86)90748-8. [DOI] [PubMed] [Google Scholar]
  3. Bastide F., Meissner G., Fleischer S., Post R. L. Similarity of the active site of phosphorylation of the adenosine triphosphatase from transport of sodium and potassium ions in kidney to that for transport of calcium ions in the sarcoplasmic reticulum of muscle. J Biol Chem. 1973 Dec 25;248(24):8385–8391. [PubMed] [Google Scholar]
  4. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  5. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  6. Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blitzer B. L., Boyer J. L. Cytochemical localization of Na+, K+-ATPase in the rat hepatocyte. J Clin Invest. 1978 Nov;62(5):1104–1108. doi: 10.1172/JCI109216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brandl C. J., Green N. M., Korczak B., MacLennan D. H. Two Ca2+ ATPase genes: homologies and mechanistic implications of deduced amino acid sequences. Cell. 1986 Feb 28;44(4):597–607. doi: 10.1016/0092-8674(86)90269-2. [DOI] [PubMed] [Google Scholar]
  9. Cantley L., Carilli C. T., Farley R. A., Perlman D. M. Location of binding sites on the (Na,K)-ATPase for fluorescein-5'-isothiocyanate and ouabain. Ann N Y Acad Sci. 1982;402:289–291. doi: 10.1111/j.1749-6632.1982.tb25749.x. [DOI] [PubMed] [Google Scholar]
  10. Capetanaki Y. G., Ngai J., Flytzanis C. N., Lazarides E. Tissue-specific expression of two mRNA species transcribed from a single vimentin gene. Cell. 1983 Dec;35(2 Pt 1):411–420. doi: 10.1016/0092-8674(83)90174-5. [DOI] [PubMed] [Google Scholar]
  11. Caplan M. J., Anderson H. C., Palade G. E., Jamieson J. D. Intracellular sorting and polarized cell surface delivery of (Na+,K+)ATPase, an endogenous component of MDCK cell basolateral plasma membranes. Cell. 1986 Aug 15;46(4):623–631. doi: 10.1016/0092-8674(86)90888-3. [DOI] [PubMed] [Google Scholar]
  12. Carroll S. L., Bergsma D. J., Schwartz R. J. Structure and complete nucleotide sequence of the chicken alpha-smooth muscle (aortic) actin gene. An actin gene which produces multiple messenger RNAs. J Biol Chem. 1986 Jul 5;261(19):8965–8976. [PubMed] [Google Scholar]
  13. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Charlemagne D., Maixent J. M., Preteseille M., Lelievre L. G. Ouabain binding sites and (Na+,K+)-ATPase activity in rat cardiac hypertrophy. Expression of the neonatal forms. J Biol Chem. 1986 Jan 5;261(1):185–189. [PubMed] [Google Scholar]
  15. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  16. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  17. Collins J. H., Zot A. S., Ball W. J., Jr, Lane L. K., Schwartz A. Tryptic digest of the alpha subunit of lamb kidney (Na+ + K+)-ATPase. Biochim Biophys Acta. 1983 Jan 26;742(2):358–365. doi: 10.1016/0167-4838(83)90322-9. [DOI] [PubMed] [Google Scholar]
  18. Emanuel J. R., Garetz S., Schneider J., Ash J. F., Benz E. J., Jr, Levenson R. Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells. Mol Cell Biol. 1986 Jul;6(7):2476–2481. doi: 10.1128/mcb.6.7.2476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Engelman D. M., Steitz T. A., Goldman A. Identifying nonpolar transbilayer helices in amino acid sequences of membrane proteins. Annu Rev Biophys Biophys Chem. 1986;15:321–353. doi: 10.1146/annurev.bb.15.060186.001541. [DOI] [PubMed] [Google Scholar]
  20. Engelman D. M., Zaccai G. Bacteriorhodopsin is an inside-out protein. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5894–5898. doi: 10.1073/pnas.77.10.5894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fambrough D. M., Bayne E. K. Multiple forms of (Na+ + K+)-ATPase in the chicken. Selective detection of the major nerve, skeletal muscle, and kidney form by a monoclonal antibody. J Biol Chem. 1983 Mar 25;258(6):3926–3935. [PubMed] [Google Scholar]
  22. Farley R. A., Ochoa G. T., Kudrow A. Location of major antibody binding domains on alpha-subunit of dog kidney Na+-K+-ATPase. Am J Physiol. 1986 Jun;250(6 Pt 1):C896–C906. doi: 10.1152/ajpcell.1986.250.6.C896. [DOI] [PubMed] [Google Scholar]
  23. Farley R. A., Tran C. M., Carilli C. T., Hawke D., Shively J. E. The amino acid sequence of a fluorescein-labeled peptide from the active site of (Na,K)-ATPase. J Biol Chem. 1984 Aug 10;259(15):9532–9535. [PubMed] [Google Scholar]
  24. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  25. Goeldner M. P., Hirth C. G., Rossi B., Ponzio G., Lazdunski M. Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer. Biochemistry. 1983 Sep 27;22(20):4685–4690. doi: 10.1021/bi00289a012. [DOI] [PubMed] [Google Scholar]
  26. Henderson R., Unwin P. N. Three-dimensional model of purple membrane obtained by electron microscopy. Nature. 1975 Sep 4;257(5521):28–32. doi: 10.1038/257028a0. [DOI] [PubMed] [Google Scholar]
  27. Hopkins B. E., Wagner H., Jr, smith T. W. Sodium- and potassium-activated adenosine triphosphatase of the nasal salt gland of the duck (Anas platyrhynchos). Purification, characterization, and NH2-terminal amino acid sequence of the phosphorylating polypeptide. J Biol Chem. 1976 Jul 25;251(14):4365–4371. [PubMed] [Google Scholar]
  28. Jørgensen P. L., Karlish S. J., Gitler C. Evidence for the organization of the transmembrane segments of (Na,K)-ATPase based on labeling lipid-embedded and surface domains of the alpha-subunit. J Biol Chem. 1982 Jul 10;257(13):7435–7442. [PubMed] [Google Scholar]
  29. Jørgensen P. L., Skriver E., Hebert H., Maunsbach A. B. Structure of the Na,K pump: crystallization of pure membrane-bound Na,K-ATPase and identification of functional domains of the alpha-subunit. Ann N Y Acad Sci. 1982;402:207–225. doi: 10.1111/j.1749-6632.1982.tb25743.x. [DOI] [PubMed] [Google Scholar]
  30. Kirley T. L., Lane L. K., Wallick E. T. Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase. J Biol Chem. 1986 Apr 5;261(10):4525–4528. [PubMed] [Google Scholar]
  31. Kreil G. Transfer of proteins across membranes. Annu Rev Biochem. 1981;50:317–348. doi: 10.1146/annurev.bi.50.070181.001533. [DOI] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. Kyte J. Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. I. Distal segment. J Cell Biol. 1976 Feb;68(2):287–303. doi: 10.1083/jcb.68.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kyte J. Immunoferritin determination of the distribution of (Na+ + K+) ATPase over the plasma membranes of renal convoluted tubules. II. Proximal segment. J Cell Biol. 1976 Feb;68(2):304–318. doi: 10.1083/jcb.68.2.304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kyte J. Molecular considerations relevant to the mechanism of active transport. Nature. 1981 Jul 16;292(5820):201–204. doi: 10.1038/292201a0. [DOI] [PubMed] [Google Scholar]
  36. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  37. Levenson R., Racaniello V., Albritton L., Housman D. Molecular cloning of the mouse ouabain-resistance gene. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1489–1493. doi: 10.1073/pnas.81.5.1489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Lytton J., Lin J. C., Guidotti G. Identification of two molecular forms of (Na+,K+)-ATPase in rat adipocytes. Relation to insulin stimulation of the enzyme. J Biol Chem. 1985 Jan 25;260(2):1177–1184. [PubMed] [Google Scholar]
  39. Lytton J. The catalytic subunits of the (Na+,K+)-ATPase alpha and alpha(+) isozymes are the products of different genes. Biochem Biophys Res Commun. 1985 Oct 30;132(2):764–769. doi: 10.1016/0006-291x(85)91198-2. [DOI] [PubMed] [Google Scholar]
  40. Macknight A. D., Leaf A. Regulation of cellular volume. Physiol Rev. 1977 Jul;57(3):510–573. doi: 10.1152/physrev.1977.57.3.510. [DOI] [PubMed] [Google Scholar]
  41. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  42. Morohashi M., Kawamura M. Solubilization and purification of Artemia salina (Na,K)-activated ATPase and NH2-terminal amino acid sequence of its larger subunit. J Biol Chem. 1984 Dec 10;259(23):14928–14934. [PubMed] [Google Scholar]
  43. Ohta T., Nagano K., Yoshida M. The active site structure of Na+/K+-transporting ATPase: location of the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and soluble peptides released by trypsin. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2071–2075. doi: 10.1073/pnas.83.7.2071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Ovchinnikov YuA, Demin V. V., Barnakov A. N., Kuzin A. P., Lunev A. V., Modyanov N. N., Dzhandzhugazyan K. N. Three-dimensional structure of (Na+ + K+)-ATPase revealed by electron microscopy of two-dimensional crystals. FEBS Lett. 1985 Oct 7;190(1):73–76. doi: 10.1016/0014-5793(85)80430-0. [DOI] [PubMed] [Google Scholar]
  45. Ovchinnikov YuA, Modyanov N. N., Broude N. E., Petrukhin K. E., Grishin A. V., Arzamazova N. M., Aldanova N. A., Monastyrskaya G. S., Sverdlov E. D. Pig kidney Na+,K+-ATPase. Primary structure and spatial organization. FEBS Lett. 1986 Jun 9;201(2):237–245. doi: 10.1016/0014-5793(86)80616-0. [DOI] [PubMed] [Google Scholar]
  46. Parnes J. R., Robinson R. R., Seidman J. G. Multiple mRNA species with distinct 3' termini are transcribed from the beta 2-microglobulin gene. 1983 Mar 31-Apr 6Nature. 302(5907):449–452. doi: 10.1038/302449a0. [DOI] [PubMed] [Google Scholar]
  47. Pauw P. G., Johnson M. D., Moore P., Morgan M., Fineman R. M., Kalka T., Ash J. F. Stable gene amplification and overexpression of sodium- and potassium-activated ATPase in HeLa cells. Mol Cell Biol. 1986 Apr;6(4):1164–1171. doi: 10.1128/mcb.6.4.1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Rozengurt E., Heppel L. A. Serum rapidly stimulates ouabain-sensitive 86-RB+ influx in quiescent 3T3 cells. Proc Natl Acad Sci U S A. 1975 Nov;72(11):4492–4495. doi: 10.1073/pnas.72.11.4492. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Schneider J. W., Mercer R. W., Caplan M., Emanuel J. R., Sweadner K. J., Benz E. J., Jr, Levenson R. Molecular cloning of rat brain Na,K-ATPase alpha-subunit cDNA. Proc Natl Acad Sci U S A. 1985 Sep;82(18):6357–6361. doi: 10.1073/pnas.82.18.6357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Serrano R., Kielland-Brandt M. C., Fink G. R. Yeast plasma membrane ATPase is essential for growth and has homology with (Na+ + K+), K+- and Ca2+-ATPases. Nature. 1986 Feb 20;319(6055):689–693. doi: 10.1038/319689a0. [DOI] [PubMed] [Google Scholar]
  53. Setzer D. R., McGrogan M., Nunberg J. H., Schimke R. T. Size heterogeneity in the 3' end of dihydrofolate reductase messenger RNAs in mouse cells. Cell. 1980 Nov;22(2 Pt 2):361–370. doi: 10.1016/0092-8674(80)90346-3. [DOI] [PubMed] [Google Scholar]
  54. Shull G. E., Greeb J., Lingrel J. B. Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain. Biochemistry. 1986 Dec 16;25(25):8125–8132. doi: 10.1021/bi00373a001. [DOI] [PubMed] [Google Scholar]
  55. Shull G. E., Schwartz A., Lingrel J. B. Amino-acid sequence of the catalytic subunit of the (Na+ + K+)ATPase deduced from a complementary DNA. Nature. 1985 Aug 22;316(6030):691–695. doi: 10.1038/316691a0. [DOI] [PubMed] [Google Scholar]
  56. Smith R. L., Macara I. G., Levenson R., Housman D., Cantley L. Evidence that a Na+/Ca2+ antiport system regulates murine erythroleukemia cell differentiation. J Biol Chem. 1982 Jan 25;257(2):773–780. [PubMed] [Google Scholar]
  57. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  58. Sweadner K. J. Enzymatic properties of separated isozymes of the Na,K-ATPase. Substrate affinities, kinetic cooperativity, and ion transport stoichiometry. J Biol Chem. 1985 Sep 25;260(21):11508–11513. [PubMed] [Google Scholar]
  59. Sweadner K. J., Gilkeson R. C. Two isozymes of the Na,K-ATPase have distinct antigenic determinants. J Biol Chem. 1985 Jul 25;260(15):9016–9022. [PubMed] [Google Scholar]
  60. Sweadner K. J., Goldin S. M. Active transport of sodium and potassium ions: mechanism, function, and regulation. N Engl J Med. 1980 Apr 3;302(14):777–783. doi: 10.1056/NEJM198004033021404. [DOI] [PubMed] [Google Scholar]
  61. Sweadner K. J. Two molecular forms of (Na+ + K+)-stimulated ATPase in brain. Separation, and difference in affinity for strophanthidin. J Biol Chem. 1979 Jul 10;254(13):6060–6067. [PubMed] [Google Scholar]
  62. Tanford C. Simple model for the chemical potential change of a transported ion in active transport. Proc Natl Acad Sci U S A. 1982 May;79(9):2882–2884. doi: 10.1073/pnas.79.9.2882. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Thomas R. C. Electrogenic sodium pump in nerve and muscle cells. Physiol Rev. 1972 Jul;52(3):563–594. doi: 10.1152/physrev.1972.52.3.563. [DOI] [PubMed] [Google Scholar]
  65. Unwin P. N., Ennis P. D. Two configurations of a channel-forming membrane protein. Nature. 1984 Feb 16;307(5952):609–613. doi: 10.1038/307609a0. [DOI] [PubMed] [Google Scholar]

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