Advertisement

The Journal of Membrane Biology

, Volume 121, Issue 2, pp 141–161 | Cite as

Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements

  • R. Bühler
  • W. Stürmer
  • H. -J. Apell
  • P. Läuger
Articles

Summary

Membrane fragments containing a high density of Na, K-ATPase can be noncovalently labeled with amphiphilic styryl dyes (e.g., RH 421). Phosphorylation of the Na,K-ATPase by ATP in the presence of Na+ and in the absence of K+ leads to a large increase of the fluorescence of RH 421 (up to 100%). In this paper evidence is presented that the styryl dye mainly responds to changes of the electric field strength in the membrane, resulting from charge movements during the pumping cycle: (i) The spectral characteristic of the ATP-induced dye response essentially agrees with the predictions for an electrochromic shift of the absorption peak. (ii) Adsorption of lipophilic anions to Na, K-ATPase membranes leads to an increase, adsorption of lipophilic cations to the decrease of dye fluorescence. These ions are known to bind to the hydrophobic interior of the membrane and to change the electric field strength in the boundary layer close to the interface. (iii) The fluorescence change that is normally observed upon phosphorylation by ATP is abolished at high concentrations of lipophilic ions. Lipophilic ions are thought to redistribute between the adsorption sites and water and to neutralize in this way the change of field strength caused by ion translocation in the pump protein. (iv) Changes of the fluorescence of RH 421 correlate with known electrogenic transitions in the pumping cycle, whereas transitions that are known to be electrically silent do not lead to fluorescence changes. The information obtained from experiments with amphiphilic styryl dyes is complementary to the results of electrophysiological investigations in which pump currents are measured as a function of transmembrane voltage. In particular, electrochromic dyes can be used for studying electrogenic processes in microsomal membrane preparations which are not amenable to electrophysiological techniques.

Key Words

Na, K-ATPase ion pumps electrogenic transport voltage-sensitive dyes electrochromic effects 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Aiuchi, T., Kobatake, Y. 1979. Electrostatic interaction between merocyamine 540 and liposomal and mitochondrial membranes.J. Membrane Biol. 45:233–244Google Scholar
  2. Altenbach, C., Seelig, J. 1985. Binding of the lipophilic cation tetraphenylphosphonium to phosphatidylcholine membranes.Biochim. Biophys. Acta 818:410–415Google Scholar
  3. Andersen, O. S., Feldberg, S., Nakadomari, H., Levy, S., McLaughlin, S. 1978. Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.Biophys. J. 21:35–70Google Scholar
  4. Apell, H.-J. 1989. Electrogenic, properties of the Na, K pump.J. Membrane Biol. 110:103–114Google Scholar
  5. Apell, H.-J., Bersch, B. 1987. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles.Biochim. Biophys. Acta 903:480–494Google Scholar
  6. Apell, H.-J., Borlinghaus, R., Läuger, P. 1987. Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents.J. Membrane Biol. 97:179–191Google Scholar
  7. Apell, H.-J., Häring, V., Roudna, M. 1990. Na,K-ATPase in artificial lipid vesicles: Comparison of Na,K and Na-only pumping mode.Biochim. Biophys. Acta 1023:81–90Google Scholar
  8. Apell, H.-J., Marcus, M.M., Anner, B.M., Oetliker, H., Läuger, P. 1985. Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase.J. Membrane Biol. 85:49–63Google Scholar
  9. Bahinski, A., Nakao, M., Gadsby, D.C. 1988. Potassium translocation by the Na/K pump is voltage insensitive.Proc. Natl. Acad. Sci. USA 85:3412–3416Google Scholar
  10. 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
  11. Benz, R., Janko, K., Läuger, P. 1976. Transport kinetics of hydrophobic ions in lipid bilayer membranes: Charge-pulse relaxation studies.Biochim. Biophys. Acta 455:701–720Google Scholar
  12. Borlinghaus, R., Apell, H.-J., Läuger, P. 1987. Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP.J. Membrane Biol. 97:161–178Google Scholar
  13. Broekhuyse, R.M. 1968. Phospholipids in tissues of the eye. I. Isolation, characterization and quantitative analysis by twodimensional thin-layer chromatography of diacyl and vinyl-ether phospholipids.Biochim. Biophys. Acta 152:307–315Google Scholar
  14. Cantley, L.C. 1981. Structure and mechanism of the (Na,K)-ATPase.Curr. Top Bioenerg. 11:201–237Google Scholar
  15. Clarke, R.J., Apell, H.-J. 1989. A stopped-flow kinetic study of the interaction of potential-sensitive oxonol dyes with lipid vesicles.Biophys. Chem. 34:225–237Google Scholar
  16. Deguchi, N., Jørgensen, P.L., Maunsbach, A.B. 1977. Ultrastructure of the sodium pump. Comparison of thin sectioning, negative staining, and freeze-fracture of purified, membranebound (Na+, K+)-ATPase.J. Cell. Biol. 75:619–634Google Scholar
  17. De Luca, M., McElroy, W.D. 1978. Purification and properties of firefly luciferase.Meth. Enzymol. 57:3–15Google Scholar
  18. Demchenko, A.P. 1986. Fluorescence analysis of protein dynamics. Essays in Biochemistry,22:120–157Google Scholar
  19. Demchenko, A.P., Ladokhin, A.S. 1988. Red-edge-excitation fluorescence spectroscopy of indole and tryptophan.Eur. Biophys. J. 15:369–379Google Scholar
  20. De Pont, J.J.H.H.M., van Prooijen-van Eeden, A., Bonting, S.L. 1978. Role of negatively charged phospholipids in highly purified (Na++K+)-ATPase from rabbit kidney outer medulla.Biochim. Biophys. Acta 508:464–477Google Scholar
  21. De Weer P. 1986. The electrogenic sodium pump: Thermodynamics and kinetics.Fortschr. Zool. 33:387–399Google Scholar
  22. De Weer, P., Gadsby, D.C., Rakowski, R.F. 1988. Voltage dependence of the Na−K pump.Annu. Rev. Physiol. 50:225–241Google Scholar
  23. Ehrenberg, B., Meiri, Z., Loew, L.M. 1984. A microsecond kinetic study of the photogenerated membrane potential of bacteriorhodopsin with a fast responding dye.Photochem. Photobiol. 39:199–205Google Scholar
  24. Ephardt, H.E., Fromherz, P. 1989. Fluorescence and photoisomerization of an amphiphilic amino-stilbazolium dye as controlled by the sensitivity of radiationless desactivation to polarity and viscosity.J. Phys. Chem. 93:7717–7725Google Scholar
  25. Ernst, A., Böhme, H., Böger, P. 1983. Phosphorylation and nitrogenase activity in isolated heterocysts fromAnabaena variabilis.Biochim. Biophys. Acta 723:83–90Google Scholar
  26. Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985. Pump currents generated by the purified Na+, K+-ATPase from kidney on black lipid membranes.EMBO J. 4:3079–3085Google Scholar
  27. Flewelling, R.F., Hubbell, W.L. 1986. The membrane dipole potential in a total membrane potential model. Applications to hydrophobic ion interactions with membranes.Biophys. J. 49:541–552Google Scholar
  28. Fluhler, E., Burnham, V.G., Loew, L.M. 1985. Spectra, membrane binding, and potentiometric responses of new charge shift probes.Biochemistry 24:5749–5755Google Scholar
  29. Forbush, B., III. 1984. Na+ movement in a single turnover of the Na pump.Proc. Natl. Acad. Sci. USA 81:5310–5314Google Scholar
  30. Gadsby, D.C., Nakao, M. 1989. Steady-state current-voltage relationship of the Na,K pump in guinea-pig ventricular myocytes.J. Gen. Physiol. 94:511–537Google Scholar
  31. Glitsch, H.G., Krahn, T., Pusch, H. 1989. The dependence of sodium pump current on internal Na concentration and membrane potential in cardioballs from sheep Purkinje fibres.Pfluegers Arch. 414:52–58Google Scholar
  32. Glynn, I.M. 1984. The electrogenic sodium pump.In: Electrogenic Transport. M.P. Blaustein and M. Lieberman, editors. pp. 33–48. Raven, New YorkGoogle Scholar
  33. Glynn, I.M. 1985. The Na, K+-transporting adenosine triphosphatase.In: The Enzymes of Biological Membranes. (2nd ed.) Vol. 3, pp. 35–114, A.N. Martonosi, editor. Plenum, New YorkGoogle Scholar
  34. Glynn, I.M., Hara, Y., Richards, D.E., Steinberg, M. 1987. Comparison of rates of cation release and of conformational change in dog kidney Na, K-ATPase.J. Physiol. 383:477–485Google Scholar
  35. Goldshlegger, R., Karlish, S.J.D., Rephaeli, A., Stein, W.D. 1987. The effect of membrane potential on the mammalian sodium-potassium pump reconstituted into phospholipid vesicles.J. Physiol. 387:331–355Google Scholar
  36. Grinvald, A., Fine, A., Farber, I.C., Hildesheim, R. 1983. Fluorescence monitoring of electrical responses from small neurons and their processes.Biophys. J. 42:195–198Google Scholar
  37. Grinvald, A., Hildesheim, R., Farber, I.C., Anglister, L. 1982. Improved fluorescent probes for the measurement of rapid changes in membrane potential.Biophys. J. 39:301–308Google Scholar
  38. Grinvald, A., Salzberg, B.M., Lev-Ram, V., Hildesheim, R. 1987. Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes.Biophys. J. 51:643–651Google Scholar
  39. Gross, D., Loew, L.M., Webb, W.W. 1986. Optical imaging of cell membrane potential changes induced by applied electric fields.Biophys. J. 50:339–348Google Scholar
  40. Heiny, J.A., Jong, D. 1990. A nonlinear electrostatic potential change in the T-system of skeletal muscle detected under passive recording conditions using potentiometric dyes.J. Gen. Physiol. 95:147–175Google Scholar
  41. Honig, B.H. 1986. Electrostatic interactions in membranes and proteins.Annu. rev. Biophys. Biophys. Chem. 15:163–193Google Scholar
  42. Hubbell, W.L. 1990. Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes.Biophys. J. 57:99–108Google Scholar
  43. Jørgensen, P.L. 1974a. Isolation of the (Na++K+)-ATPase.Methods Enzymol. 32:277–290Google Scholar
  44. Jørgensen, P.L. 1974b. Purification and characterization of (Na++K+)-ATPase: III. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate.Biochim. Biophys. Acta 356:36–52Google Scholar
  45. Jørgensen, P.L. 1982. Mechanism of the Na+, K+ pump. Protein structure and conformations of the purified (Na++K+)-ATPase.Biochim. Biophys. Acta 694:27–68Google Scholar
  46. Jørgensen, P.L., Andersen, J.P. 1988. Structural basis for E1−E2 conformational transitions in Na, K-pump and Ca-pump, proteins.J. Membrane Biol. 103:95–120Google Scholar
  47. Kapakos, J.G., Steinberg, M. 1982. Fluorescent labeling of (Na+−K+)-ATPase by 5-iodoacetamidofluorescein.Biochim. Biophys. Acta 693:493–496Google Scholar
  48. Kapakos, J.G., Steinberg, M. 1986a. Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein.J. Biol. Chem. 261:2084–2089Google Scholar
  49. Kapakos, J.G., Steinberg, M. 1986b. 5-lodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover.J. Biol. Chem. 261:2090–2096Google Scholar
  50. Kaplan, J.H., Forbush, B., III, Hoffman, J.F. 1978. Rapid photolytic release of adenosine-5′-triphosphate from a protected analogue: Utilization by the Na:K pump of human red blood cell ghosts.Biochemistry 17:1929–1935Google Scholar
  51. Klodos, I., Forbush, B., III, 1988. Rapid conformational changes of the Na/K pump revealed by a fluorescent dye, RH-160.J. Gen. Physiol. 92:46a (abstr.)Google Scholar
  52. Krasne, S. 1983. Interactions of voltage-sensing dyes with membranes: III Electrical properties induced by merocyamine 540.Biophys. J. 44:305–314Google Scholar
  53. Läuger, P., Benz, R., Stark, G., Bamberg, E., Jordan, P.C., Fahr, A., Brock, W. 1981. Relaxation studies of ion transport systems in lipid bilayer membranes.Q. Rev. Biophys. 14:513–598Google Scholar
  54. Loew, L.M. 1982. Design and characterization of electrochromic membrane probes.J. Biochem. Biophys. Methods 6:243–260Google Scholar
  55. Loew, L.M., Scully, S., Simpson, L., Waggoner, A.S. 1979. Evidence for a charge-shift electrochromic mechanism in a probe of membrane potential.Nature 281:497–499Google Scholar
  56. Loew, L.M., Simpson, L.L., 1981. Charge-shift probes of membrane potential. A probable electrochromic mechanism for aminostyrylpyridinium probes on a hemispherical lipid bilayer.Biophys. J. 34:353–365Google Scholar
  57. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagents.J. Biol. Chem. 193:265–275Google Scholar
  58. 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. Zabler, editors. pp. 209–219. Springer, BerlinGoogle Scholar
  59. Mårdh, S., Zetterquist, Ö. 1974. Phosphorylation and dephosphorylation reactions of bovine brain (Na++K+)-stimulated ATP phosphohydrolase studied by a rapid-mixing technique.Biochim. Biophys. Acta 350:473–483Google Scholar
  60. Markin, V.S., Grigor'ev, P.A., Yermishkin, L.N. 1971. Forward passage of ions across lipid membranes: I. Mathematical model.Biofizika 16:1011–1018Google Scholar
  61. McCray, J.A., Herbette, L., Kihara, T., Trentham, D.R. 1980. A new approach to time-resolved studies of ATP-requiring biological systems; Laserflash photolysis of caged ATP.Proc. Natl. Acad. Sci. USA 77:7237–7241Google Scholar
  62. McLaughlin, S. 1977 Electrostatic potentials at membrane-solution interfaces.Curr. Top. Membr. Transp. 9:71–144Google Scholar
  63. McLaughlin, S. 1989. The electrostatic properties of membranes.Annu. Rev. Biophys. Biophys. Chem. 18:113–136Google Scholar
  64. Müller, W., Windisch, H., Tritthart, H.A. 1986. Fluorescent styryl dyes applied as fast optical probes of cardiac action potential.Eur. Biophys. J. 14:103–111Google Scholar
  65. Nagel, G., Slayman, C., Klodos, I. 1989. Fluorescence probing of a major conformational change in the plasma membrane H-ATPase ofNeurospora.Biophys. J. 55:338a Google Scholar
  66. Nakao, M., Gadsby, D.C. 1989. [Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea-pig ventricular myocytes.J. Gen. Physiol 94:539–565Google Scholar
  67. Peters, W.H.M., Fleuren-Jakobs, A.M.M., de Pont, J.J.H.H.M., Bonting, S.L. 1981. Studies on (Na++K)-activated ATPase: XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation.Biochim. Biophys. Acta 649:541–549Google Scholar
  68. Pickar, A.D., Benz, R. 1987. Transport of oppositely charged lipophilic probe ions in lipid bilayer membranes having various structures.J. Membrane Biol. 44:353–376Google Scholar
  69. rakowski, R.F., Gadsby, D.C., De Weer, P. 1989. Stoichiometry and voltage dependence of the sodium pump in voltagechamped, internally dialyzed squid axon.J. Gen. Physiol. 93:903–941Google Scholar
  70. Rakowski, R.F., Paxson, C.L. 1988. Voltage dependence of Na/K pump current inXenopus oocytes.J. Membrane Biol. 106:173–182Google Scholar
  71. Rey, H.G., Moosmayer, M., Anner, B.M. 1987. Characterization of (Na++K+)-ATPase-liposomes: III. Controlled activation and inhibition of symmetric pumps by timed asymmetric ATP, RbCl, and cardiac glycoside addition.Biochim. Biophys. Acta 900:27–37Google Scholar
  72. Schuurmans Stekhoven, F.M.A.H., Swarts, H.G.P., 't Lam, G.K., Zou, Y.S., De Pont, J.J.H.H.M. 1988. Phosphorylation of (Na−K+)-ATPase; stimulation and inhibition by substituted and unsubstituted amines.Biochim. Biophys. Acta 937:161–176Google Scholar
  73. 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
  74. Schweigert, B., Lafaire, A.V., Schwarz, W. 1988. Voltage dependence of the Na,K-ATPase: Measurements of ouabain-dependent membrane current and ouabain binding in oocytes ofXenopus laevis.Pfluegers Arch. 412:579–588Google Scholar
  75. Steinberg, M., Karlish, S.J.D. 1989. Studies on conformational changes in Na, K-ATPase labeled with 5-iodoacetamidofluorescein.J. Biol. Chem. 264:2726–2734Google Scholar
  76. Stürmer, W., Apell, H.-J., Wuddel, I., Läuger, P. 1989. Conformational transitions and charge translocation by the Na,K pump: Comparison of optical and electrical transients elicited by ATP-concentration jumps.J. Membrane Biol. 110:67–86Google Scholar
  77. Stürmer, W., Bühler, R., Apell, H.-J., Läuger, P. 1991. Charge translocation by the Na, K-pump: II. Ion binding and release at the extracellular side.J. Membrane Biol. 121:163–176Google Scholar
  78. Szabo, G. 1974. Dual mechanism of the action of cholesterol on membrane permeability.Nature 252:47–49Google Scholar
  79. Taniguchi, K., Post, R.L. 1975. Synthesis of adenosine triphosphate and exchange between inorganic phosphate and adenosine triphosphate in sodium and potassium ion transport adenosine triphosphatase.J. Biol. Chem. 250:3010–3018Google Scholar
  80. Thorne, S.W., Duniec, J.T. 1983. The physical principles of energy transduction in chloroplast thylakoid membranes.Q. Rev. Biophys. 16:197–278Google Scholar
  81. Tyson, P.A., Steinberg, M., Wallick, E.T., Kirley, T.L. 1989. Identification of the 5-iodoacetamidofluorescein reporter site on the Na,K-ATPase.J. Biol. Chem. 264:726–734Google Scholar
  82. Waggoner, A.S., Grinvald, A. 1977. Mechanism of rapid optical changes of potential sensitive dyes.Ann. N.Y. Acad. Sci. 303:217–241Google Scholar
  83. Zimányi, L., Garab, G., 1989. Configuration of the electric field and distribution of ions in energy transducing biological membranes: Model calculations in a vesicle containing discrete charges.J. Theor. Biol. 138:59–76Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1991

Authors and Affiliations

  • R. Bühler
    • 1
  • W. Stürmer
    • 1
  • H. -J. Apell
    • 1
  • P. Läuger
    • 1
  1. 1.Department of BiologyUniversity of KonstanzKonstanzFederal Republic of Germany

Personalised recommendations