Skip to main content
SpringerLink
Log in

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

  • Articles
  • Published:
The Journal of Membrane Biology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Aiuchi, T., Kobatake, Y. 1979. Electrostatic interaction between merocyamine 540 and liposomal and mitochondrial membranes.J. Membrane Biol. 45:233–244

    Google Scholar 

  • Altenbach, C., Seelig, J. 1985. Binding of the lipophilic cation tetraphenylphosphonium to phosphatidylcholine membranes.Biochim. Biophys. Acta 818:410–415

    Google Scholar 

  • Andersen, O. S., Feldberg, S., Nakadomari, H., Levy, S., McLaughlin, S. 1978. Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.Biophys. J. 21:35–70

    Google Scholar 

  • Apell, H.-J. 1989. Electrogenic, properties of the Na, K pump.J. Membrane Biol. 110:103–114

    Google Scholar 

  • Apell, H.-J., Bersch, B. 1987. Oxonol VI as an optical indicator for membrane potentials in lipid vesicles.Biochim. Biophys. Acta 903:480–494

    Google Scholar 

  • 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. 97:179–191

    Google Scholar 

  • 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 1023:81–90

    Google Scholar 

  • 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. 85:49–63

    Google Scholar 

  • Bahinski, A., Nakao, M., Gadsby, D.C. 1988. Potassium translocation by the Na/K pump is voltage insensitive.Proc. Natl. Acad. Sci. USA 85:3412–3416

    Google Scholar 

  • Beeler, T.J., Farmen, R.H., Martonosi, A.N. 1981. The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.J. Membrane Biol. 62:113–137

    Google Scholar 

  • Benz, R., Janko, K., Läuger, P. 1976. Transport kinetics of hydrophobic ions in lipid bilayer membranes: Charge-pulse relaxation studies.Biochim. Biophys. Acta 455:701–720

    Google Scholar 

  • 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. 97:161–178

    Google Scholar 

  • 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 152:307–315

    Google Scholar 

  • Cantley, L.C. 1981. Structure and mechanism of the (Na,K)-ATPase.Curr. Top Bioenerg. 11:201–237

    Google Scholar 

  • 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. 34:225–237

    Google Scholar 

  • 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. 75:619–634

    Google Scholar 

  • De Luca, M., McElroy, W.D. 1978. Purification and properties of firefly luciferase.Meth. Enzymol. 57:3–15

    Google Scholar 

  • Demchenko, A.P. 1986. Fluorescence analysis of protein dynamics. Essays in Biochemistry,22:120–157

    Google Scholar 

  • Demchenko, A.P., Ladokhin, A.S. 1988. Red-edge-excitation fluorescence spectroscopy of indole and tryptophan.Eur. Biophys. J. 15:369–379

    Google Scholar 

  • 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 508:464–477

    Google Scholar 

  • De Weer P. 1986. The electrogenic sodium pump: Thermodynamics and kinetics.Fortschr. Zool. 33:387–399

    Google Scholar 

  • De Weer, P., Gadsby, D.C., Rakowski, R.F. 1988. Voltage dependence of the Na−K pump.Annu. Rev. Physiol. 50:225–241

    Google Scholar 

  • 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. 39:199–205

    Google Scholar 

  • 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. 93:7717–7725

    Google Scholar 

  • Ernst, A., Böhme, H., Böger, P. 1983. Phosphorylation and nitrogenase activity in isolated heterocysts fromAnabaena variabilis.Biochim. Biophys. Acta 723:83–90

    Google Scholar 

  • 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. 4:3079–3085

    Google Scholar 

  • 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. 49:541–552

    Google Scholar 

  • Fluhler, E., Burnham, V.G., Loew, L.M. 1985. Spectra, membrane binding, and potentiometric responses of new charge shift probes.Biochemistry 24:5749–5755

    Google Scholar 

  • Forbush, B., III. 1984. Na+ movement in a single turnover of the Na pump.Proc. Natl. Acad. Sci. USA 81:5310–5314

    Google Scholar 

  • Gadsby, D.C., Nakao, M. 1989. Steady-state current-voltage relationship of the Na,K pump in guinea-pig ventricular myocytes.J. Gen. Physiol. 94:511–537

    Google Scholar 

  • 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. 414:52–58

    Google Scholar 

  • Glynn, I.M. 1984. The electrogenic sodium pump.In: Electrogenic Transport. M.P. Blaustein and M. Lieberman, editors. pp. 33–48. Raven, New York

    Google Scholar 

  • 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 York

    Google Scholar 

  • 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. 383:477–485

    Google Scholar 

  • 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. 387:331–355

    Google Scholar 

  • Grinvald, A., Fine, A., Farber, I.C., Hildesheim, R. 1983. Fluorescence monitoring of electrical responses from small neurons and their processes.Biophys. J. 42:195–198

    Google Scholar 

  • Grinvald, A., Hildesheim, R., Farber, I.C., Anglister, L. 1982. Improved fluorescent probes for the measurement of rapid changes in membrane potential.Biophys. J. 39:301–308

    Google Scholar 

  • 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. 51:643–651

    Google Scholar 

  • Gross, D., Loew, L.M., Webb, W.W. 1986. Optical imaging of cell membrane potential changes induced by applied electric fields.Biophys. J. 50:339–348

    Google Scholar 

  • 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. 95:147–175

    Google Scholar 

  • Honig, B.H. 1986. Electrostatic interactions in membranes and proteins.Annu. rev. Biophys. Biophys. Chem. 15:163–193

    Google Scholar 

  • Hubbell, W.L. 1990. Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes.Biophys. J. 57:99–108

    Google Scholar 

  • Jørgensen, P.L. 1974a. Isolation of the (Na++K+)-ATPase.Methods Enzymol. 32:277–290

    Google Scholar 

  • 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 356:36–52

    Google Scholar 

  • Jørgensen, P.L. 1982. Mechanism of the Na+, K+ pump. Protein structure and conformations of the purified (Na++K+)-ATPase.Biochim. Biophys. Acta 694:27–68

    Google Scholar 

  • 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. 103:95–120

    Google Scholar 

  • Kapakos, J.G., Steinberg, M. 1982. Fluorescent labeling of (Na+−K+)-ATPase by 5-iodoacetamidofluorescein.Biochim. Biophys. Acta 693:493–496

    Google Scholar 

  • Kapakos, J.G., Steinberg, M. 1986a. Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluorescein.J. Biol. Chem. 261:2084–2089

    Google Scholar 

  • Kapakos, J.G., Steinberg, M. 1986b. 5-lodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover.J. Biol. Chem. 261:2090–2096

    Google Scholar 

  • 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 17:1929–1935

    Google Scholar 

  • 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 

  • Krasne, S. 1983. Interactions of voltage-sensing dyes with membranes: III Electrical properties induced by merocyamine 540.Biophys. J. 44:305–314

    Google Scholar 

  • 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. 14:513–598

    Google Scholar 

  • Loew, L.M. 1982. Design and characterization of electrochromic membrane probes.J. Biochem. Biophys. Methods 6:243–260

    Google Scholar 

  • 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 281:497–499

    Google Scholar 

  • 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. 34:353–365

    Google Scholar 

  • Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagents.J. Biol. Chem. 193:265–275

    Google Scholar 

  • 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, Berlin

    Google Scholar 

  • 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 350:473–483

    Google Scholar 

  • Markin, V.S., Grigor'ev, P.A., Yermishkin, L.N. 1971. Forward passage of ions across lipid membranes: I. Mathematical model.Biofizika 16:1011–1018

    Google Scholar 

  • 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 77:7237–7241

    Google Scholar 

  • McLaughlin, S. 1977 Electrostatic potentials at membrane-solution interfaces.Curr. Top. Membr. Transp. 9:71–144

    Google Scholar 

  • McLaughlin, S. 1989. The electrostatic properties of membranes.Annu. Rev. Biophys. Biophys. Chem. 18:113–136

    Google Scholar 

  • Müller, W., Windisch, H., Tritthart, H.A. 1986. Fluorescent styryl dyes applied as fast optical probes of cardiac action potential.Eur. Biophys. J. 14:103–111

    Google Scholar 

  • 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 

  • 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 94:539–565

    Google Scholar 

  • 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 649:541–549

    Google Scholar 

  • Pickar, A.D., Benz, R. 1987. Transport of oppositely charged lipophilic probe ions in lipid bilayer membranes having various structures.J. Membrane Biol. 44:353–376

    Google Scholar 

  • 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. 93:903–941

    Google Scholar 

  • Rakowski, R.F., Paxson, C.L. 1988. Voltage dependence of Na/K pump current inXenopus oocytes.J. Membrane Biol. 106:173–182

    Google Scholar 

  • 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 900:27–37

    Google Scholar 

  • 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 937:161–176

    Google Scholar 

  • Schwartz, A., Nagano, K., Nakao, M., Lindenmayer, G.E., Allen, J.C. 1971. The sodium- and potassium-activated adenosinetriphosphatase system.Meth. Pharmacol. 1:361–388

    Google Scholar 

  • 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. 412:579–588

    Google Scholar 

  • Steinberg, M., Karlish, S.J.D. 1989. Studies on conformational changes in Na, K-ATPase labeled with 5-iodoacetamidofluorescein.J. Biol. Chem. 264:2726–2734

    Google Scholar 

  • 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. 110:67–86

    Google Scholar 

  • 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. 121:163–176

    Google Scholar 

  • Szabo, G. 1974. Dual mechanism of the action of cholesterol on membrane permeability.Nature 252:47–49

    Google Scholar 

  • 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. 250:3010–3018

    Google Scholar 

  • Thorne, S.W., Duniec, J.T. 1983. The physical principles of energy transduction in chloroplast thylakoid membranes.Q. Rev. Biophys. 16:197–278

    Google Scholar 

  • 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. 264:726–734

    Google Scholar 

  • Waggoner, A.S., Grinvald, A. 1977. Mechanism of rapid optical changes of potential sensitive dyes.Ann. N.Y. Acad. Sci. 303:217–241

    Google Scholar 

  • 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. 138:59–76

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biology, University of Konstanz, D-7750, Konstanz, Federal Republic of Germany

    R. Bühler, W. Stürmer, H. -J. Apell & P. Läuger

Authors
  1. R. Bühler
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. W. Stürmer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. -J. Apell
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P. Läuger
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Deceased (September 13, 1990).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Bühler, R., Stürmer, W., Apell, H.J. et al. Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements. J. Membrain Biol. 121, 141–161 (1991). https://doi.org/10.1007/BF01870529

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01870529

Key Words

Search

Navigation