The Journal of Membrane Biology

, Volume 85, Issue 1, pp 49–63 | Cite as

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase

  • H. -J. Apell
  • M. M. Marcus
  • B. M. Anner
  • H. Oetliker
  • H. Oetliker
  • P. Läuger


A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as a fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.

Key Words

Na,K-ATPase reconstitution potential sensitive dye ion fluxes transport kinetics activation energy 


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  1. Abercrombie, R., De Weer, P. 1978. Electric current generated by squid axon: External K and internal ADP effects.Am. J. Physiol. 44:389–400Google Scholar
  2. Abeywardena, M.Y., Allen, Th.M., Charnock, J.S. 1983. Lipid-protein interactions of reconstituted membrane-associated adenosine triphosphatases. Use of a gel-filtration procedure to examine phospholipid-activity relationships.Biochim. Biophys. Acta 729:62–74PubMedGoogle Scholar
  3. Anner, B.M. 1980. Ratio of Na: K transport in reconstituted sodium pump vesicles.Biochem. Biophys. Res. Commun. 94:1233–1241PubMedGoogle Scholar
  4. Anner, B.M. 1981. A K-selective cation channel formed by Na,K-ATPase in liposomes.Biochem. Int. 2:365–371Google Scholar
  5. Anner, B.M., Lane, L.K., Schwartz, A., Pitts, B.J.R. 1977. A reconstituted Na++K+ pump in liposomes containing purified (Na++K+)-ATPase from kidney medulla.Biochim. Biophys. Acta 467:340–345PubMedGoogle Scholar
  6. Anner, B.M., Marcus, M.M., Moosmayer, M. 1984a. Reconstitution of Na,K-ATPase.In: Enzymes, Receptors and Carriers of Biomembranes. pp. 81–96. Springer-Verlag, HeidelbergGoogle Scholar
  7. Anner, B.M., Moosmayer, M. 1981. Preparation of Na,K-ATPase-containing liposomes with predictable transport properties by a procedure relating the Na,K-transport capacity to the ATPase activity.J. Biochem. Biophys. Methods 5:299–306Google Scholar
  8. Anner, B.M., Moosmayer, M. 1982. On the kinetics of the Na:K exchange in the initial and final phase of sodium pump activity in liposomes.J. Membr. Sci. 11:27–37Google Scholar
  9. Anner, B.M., Robertson, J.D., Ting-Beall, H.P. 1984b. Characterization of (Na++K+)-ATPase liposomes. I. Effect of enzyme concentration and modification of liposome size, intramembrane particle formation and Na+,K+-transport.Biochim. Biophys. Acta 773:253–261PubMedGoogle Scholar
  10. Bartlett, G. 1959. Phosphorous assay in column chromatography.J. Biol. Chem. 234:466–468PubMedGoogle Scholar
  11. Beeler, T.J., Farmen R.H., Martonosi, A.N. 1981. The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.J. Membrane Biol. 62:113–137Google Scholar
  12. Benz, R., Stark, G., Janko, K., Läuger, P. 1973. Valinomycin-mediated ion transport through neutral lipid membranes: Influence of carbon chain length and temperature.J. Membrane Biol. 14:339–364Google Scholar
  13. Brotherus, J.R., Jacobsen, L., Jørgensen, P.L. 1983. Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units. Preparation, assay and reconstitution of active Na+,K+ transport.Biochim. Biophys. Acta 731:290–303PubMedGoogle Scholar
  14. Cantley, L.C. 1981. Structure and mechanism of the (Na,K)-ATPase.Curr. Top. Bioenerg. 11:201–237Google Scholar
  15. Carruthers, A., Melchior, D.L. 1983. Studies of the relationship between bilayer water permeability and bilayer physical state.Biochemistry 22:5797–5807Google Scholar
  16. Cornelius, F., Skou, J.C. 1984. Reconstitution of (Na++K+)-ATPase into phospholipid vesicles with full recovery of its specific activity.Biochim. Biophys. Acta 772:357–373PubMedGoogle Scholar
  17. Dixon, J.F., Hokin, L.E. 1980. The reconstituted (Na,K)-ATPase is electrogenic.J. Biol. Chem. 255:10681–10686PubMedGoogle Scholar
  18. Forbush, B., III, 1984. An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins.Anal. Biochem. (in press) Google Scholar
  19. Forgac, M., Chin, G. 1982. Na+ transport by the (Na+)-stimulated adenosine triphosphatase.J. Biol. Chem. 257:5652–5655PubMedGoogle Scholar
  20. Glitsch, H.G. 1982. Electrogenic Na pumping in the heart.Annu. Rev. Physiol. 44:389–400Google Scholar
  21. Goldin, S.M., Tong, S.W. 1974. Reconstitution of active transport catalyzed by the purified sodium and potassium ion-stimulated adenosine triphosphatase from canine renal medulla.J. Biol. Chem. 249:5907–5915PubMedGoogle Scholar
  22. Goldman, D.E. 1943. Potential, impedance, and rectification in membranes.J. Gen. Physiol. 27:37–60CrossRefGoogle Scholar
  23. Hilden, S., Rhee, H.M., Hokin, L.E. 1974. Sodium transport by phospholipid vesicles containing purified sodium and potassium ion-activated adenosine triphosphatase.J. Biol. Chem. 249:7432–7440PubMedGoogle Scholar
  24. Hoffman, J.F., Kaplan, J.H., Callahan, T.J. 1979. The Na:K pump in red cells is electrogenic.Fed. Proc. 38:2440–2441PubMedGoogle Scholar
  25. Hoffman, J.F., Laris, P.C. 1974. Determination of membrane potentials in human andAmphiuma red blood cells by means of a fluorescent probe.J. Physiol. (London) 239:519–552Google Scholar
  26. Jackson, R.L., Verkleij, A.J., Zoelen, E.J.J. van, Lane, L.K., Schwartz, A., Deenen, L.L.M. van 1980. Asymmetric incorporation of Na+,K+-ATPase into phospholipid vesicles.Arch. Biochem. Biophys. 200:269–278PubMedGoogle Scholar
  27. Jørgensen, P.L. 1974. Isolation of (Na++K+)-ATPase.Methods Enzymol. 32:277–290PubMedGoogle Scholar
  28. Jørgensen, P.L. 1982. Mechanism of the Na+,K+ pump. Protein structure and conformations of the pure (Na++K+)-ATPase.Biochim. Biophys. Acta 694:27–68PubMedGoogle Scholar
  29. Karlish, S.J.D., Lieb, W.R., Stein, W.D. 1982. Combined effects of ATP and phosphate on rubidium exchange mediated by Na−K-ATPase reconstituted into phospholipid vesicles.J. Physiol. (London) 328:333–350Google Scholar
  30. Karlish, S.J.D., Pick, U. 1981. Sidedness of the effects of sodium and potassium ions on the conformational state of the sodium-potassium pump.J. Physiol. (London) 312:505–529Google Scholar
  31. Krasne, S. 1983. Interactions of voltage-sensing dyes with membranes. III. Electrical properties induced by merocyanine 540.Biophys. J. 44:305–314PubMedGoogle Scholar
  32. Läuger, P., Stark, G. 1970. Kinetics of carrier-mediated ion transport across lipid bilayer membranes.Biochim. Biophys. Acta 211:458–466PubMedGoogle Scholar
  33. Lawaczeck, R. 1979. On the permeability of water molecules across vesicular lipid bilayers.J. Membrane Biol. 51:229–261Google Scholar
  34. Lederer, W.J., Nelson, M.T. 1984. Sodium pump stoichiometry determined by simultaneous measurements of sodium efflux and membrane current in barnacle.J. Physiol. (London) 348:665–677Google Scholar
  35. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagents.J. Biol. Chem. 193:265–275PubMedGoogle Scholar
  36. Lüdi, H., Oetliker, H., Brodbeck, U. 1981. Use of a potentiometric cyanine dye in the study of reconstituted membrane proteins.In. Membrane Proteins. A. Azzi, U. Brodbeck, and P. Zahler, editors. pp. 209–219. Springer, BerlinGoogle Scholar
  37. Lüdi, H., Oetliker, H., Brodbeck, U., Ott, P., Schwendimann, B., Fulpius, B.W. 1983. Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye.J. Membrane Biol. 74:75–84Google Scholar
  38. McLaughlin, S.A. 1977. Electrostatic potentials at membrane-solution interfaces.Curr. Top. Membr. Transp. 9:71–144Google Scholar
  39. Milsmann, M.H.W., Schwendener, R.A., Weder, H.G. 1978. The preparation of large single bilayer liposomes by a fast and controlled dialysis.Biochim. Biophys. Acta 512:147–155PubMedGoogle Scholar
  40. Oetliker, H. 1980. Studies on the mechanism causing optical excitation-contraction coupling signals in skeletal muscle.J. Physiol. (London) 305:26–32Google Scholar
  41. Oetliker, H. 1981. Divalent cation concentration-dependent fluorescence of isolated sarcoplasmic reticulum vesicles stained with indodicarbocyanine.J. Physiol. (London) 318:11–12Google Scholar
  42. Oetliker, H. 1982. An appraisal of the evidence for a sarcoplasmic reticulum membrane potential and its relation to calcium release in skeletal muscle.J. Muscle Res. Cell Motil. 3:247–272PubMedGoogle Scholar
  43. Post, R.L., Sen, A.K., Rosenthal A.S. 1965. A phosphorylated intermediate in adenosine triphosphate-dependent sodium and potassium transport across kidney membranes.J. Biol. Chem. 240:1437–1444PubMedGoogle Scholar
  44. Racker, E., Fisher, L.W. 1975. Reconstitution of an ATP-dependent sodium pump with an ATPase from electric eel and pure phospholipids.Biochem. Biophys. Res. Commun. 67:1144–1150PubMedGoogle Scholar
  45. Rhoden, V., Goldin, S.M. 1979. Formation of unilamellar lipid vesicles of controllable dimensions by detergent dialysis.Biochemistry 18:4173–4176PubMedGoogle Scholar
  46. Robinson, J.D. 1983. Kinetic analyses and the reaction mechanism of the Na,K-ATPase.In: Structure, Mechanism and Function of the Na/K Pump. J. F. Hoffman and B. Forbush. III, editors.Curr. Top. Membr. Transp. 19:485–512Google Scholar
  47. Robinson, J.D., Flashner, M.S. 1979. The (Na++K+)-activated ATPase. Enzymatic and transport properties.Biochim. Biophys. Acta 549:145–176PubMedGoogle Scholar
  48. Ross, W.N., Salzberg, B.M., Cohen, L.B., Grinvald, A., Davila, H.V., Waggoner, A.S., Wang, C.H. 1977. Changes in absorption, fluorescence, dichroism, and birefringence in stained giant axons: Optical measurements of membrane potential.J. Membrane Biol. 33:141–183Google Scholar
  49. Schuurmans-Stekhoven, F., Bonting, S.L. 1981. Transport of adenosin triphosphatases: Properties and function.Physiol. Rev. 61:1–76Google Scholar
  50. Schwartz, A., Nagano, K., Nakao, M., Lindenmayer, G.E., Allen, J.C. 1971. The sodium-and potassium-activated adenosinetriphosphatase system.Meth. Pharmacol. 1:361–388Google Scholar
  51. Sims, P.J., Waggoner, A.S., Wang, Ch.H., Hoffman, J.F. 1974. Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles.Biochemistry 13:3315–3329PubMedGoogle Scholar
  52. Skou, J.C. 1975. The (Na++K+) activated enzyme system and its relationship to transport of sodium and potassium.Q. Rev. Biophys. 7:401–431Google Scholar
  53. Skriver, E., Maunsbach, A.B., Anner, B.M., Jørgensen, P.L. 1980a. Electron microscopy of phospholipid vesicles reconstituted with purified renal Na,K-ATPase.Cell Biol. Int. Rep. 4:585–591PubMedGoogle Scholar
  54. Skriver, E., Maunsbach, A.B., Jørgensen, P.L. 1980b. Ultrastructure of Na,K-transport vesicles reconstituted with purified renal Na,K-ATPase.J. Cell. Biol. 86:746–754PubMedGoogle Scholar
  55. Stark, G., Benz, R. 1971. The transport of potassium through lipid bilayer membranes by the neutral carriers valinomycin and monactin. Experimental studies to a previously proposed model.J. Membrane Biol. 5:133–153Google Scholar
  56. Waggoner, A.S. 1979. Dye indicators of membrane potential.Annu. Rev. Biophys. Bioeng. 8:47–68PubMedGoogle Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • H. -J. Apell
    • 1
  • M. M. Marcus
    • 1
  • B. M. Anner
    • 2
  • H. Oetliker
    • 3
  • H. Oetliker
    • 3
  • P. Läuger
    • 1
  1. 1.Fakultät für BiologieUniversität KonstanzKonstanzFederal Republic of Germany
  2. 2.Département de Pharmacologie, Centre Médical UniversitaireUniversité de GenèveGenève 4Switzerland
  3. 3.Physiologisches Institut der Universität BernBernSwitzerland

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