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The Journal of Membrane Biology

, Volume 145, Issue 2, pp 165–173 | Cite as

Sequential potassium binding at the extracellular side of the Na,K-Pump

  • R. Bühler
  • H. -J. Apell
Articles

Abstract

Ion binding at the extracellular face of the Na,K-ATPase is electrogenic and can be monitored by the styryl dye RH 421 in membrane fragments containing a high density of the Na,K-pumps. The fluorescent probe is noncovalently bound to the membrane and responds to changes of the local electric field generated by binding or release of cations inside the protein. Due to the fact that K+ binding from the extracellular side is an electrogenic reaction, it is possible to detect the amount of ions bound to the pump as function of the aqueous concentration. The results are in contradiction to a second order reaction, i.e., a simultaneous binding of two K+ ions. A mathematical model is presented to discuss the nature of the two step binding process. On the basis of this model the data allow a quantitative distinction between binding of the first and the second K+ ion. The temperature dependence of ion binding has been investigated. At low temperatures the apparent dissociation constants differ significantly. In the temperature range above 20°C the resulting apparent dissociation constants for both K+ ions merge and have values between 0.2 and 0.3 mm, which is consistent with previous experiments. The activation energy for the half saturating concentration of K+ is 22 kJ/mol. Additional analysis of the titration curve of K+ binding to the state P — E2 by the Hill equation yields a Hill coefficient, nHill, of 1.33, which is in agreement with previously published data.

Key words

Na,K-ATPase Potassium binding Electrogenic transport Cation binding site Sequential binding Activation energy 

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References

  1. Apell, H.-J. 1989. Electrogenic properties of the Na,K-pump. J. Membrane Biol. 110:103–114Google Scholar
  2. Apell, H.-J., Nelson, M.T., Marcus, M.M., Läuger, P. 1986. Effects of ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump. Biochim. Biophys. Acta 857:105–115Google Scholar
  3. Bühler, R., Stürmer, W., Apell, H.-J., Läuger, P. 1991. Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements. J. Membrane Biol. 121:141–161Google Scholar
  4. Clarke, R.P., Schrimpf, P., Schöneich, M. 1992. Spectroscopic investigations of the potential-sensitive membrane probe RH 421. Biochim. Biophys. Acta 1112:142–152Google Scholar
  5. Forbush III, B. 1987. Rapid release of 42K and 86Rb from an occluded state of the Na,K-pump in the presence of ATP or ADP. J. Biol. Chem. 262:11116–11127Google Scholar
  6. Forbush III, B. 1988. Occluded ions and Na,K-ATPase. In: The Na+,K+-Pump, Part A: Molecular Aspects. J.C. Skou, J.G. Nørby, A.B. Maunsbach and M. Esmann, editors. Prog. Clin. Biol. Res. 268A, pp. 229–248. A.R. Liss, New York.Google Scholar
  7. Gache, C., Rossi, B., Lazdunski, M. 1967. (Na+,K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Eur. J. Biochem. 65:293–306Google Scholar
  8. Glynn, I.M. 1985. The Na+,K+-transporting adenosine triphosphatase. In: The Enzymes of Biological Membranes. A.N. Martonosi, editor. Vol. 3 (2nd.), pp. 35–114. Plenum, New YorkGoogle Scholar
  9. Glynn, I.M., Richards, D.E. 1982. Occlusion of rubidium ions by the sodium-potassium pump: Its implications for the mechanism of potassium transport. J. Physiol. 330:17–43.Google Scholar
  10. Grinvald, A., Hildesheimer, R., Farber, I.C., Anglister, L. 1982. Improved fluorescent probes for the measurements of rapid changes in membrane potential. Biophys. J. 39:301–308Google Scholar
  11. Heyse, S., Wuddel, I., Apell, H.-J., Stürmer, W. 1994. Partial reactions of the Na,K-ATPase: Determination of rate constants. J. Gen. Physiol. 104:197–240Google Scholar
  12. Jørgensen, P.L. 1974. Isolation of the (Na+ + K+)-ATPase. Methods Enzymol. 32:277–290Google Scholar
  13. Karlish, S.J.D., Stein, W.D. 1982. Passive rubidium fluxes mediated by Na,K-ATPase reconstituted into phospholipid vesicles when ATP-and phosphate-free. J. Physiol. 328:295–316Google Scholar
  14. Läuger, P. 1991. In: Electrogenic Ion Pumps. pp. 168–224. Sinauer, Sutherland, MAGoogle Scholar
  15. Läuger, P., Apell, H.-J. 1986. A microscopic model for the currentvoltage behavior of the Na,K-pump. Eur. Biophys. J. 13:309–321Google Scholar
  16. Lowry, O.H., Rosenbrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurements with the folin phenol reagents. J. Biol. Chem. 193:265–275PubMedGoogle Scholar
  17. Marcus, M.M., Apell, H.-J., Roudna, M., Schwendener, R.A., Weder, H.-G. and Läuger, P. 1986. (Na+ + K+)-ATPase in artificial lipid vesicles: influence of lipid structure on pumping rate. Biochim. Biophys. Acta 845:270–278Google Scholar
  18. Rakowski, R.F., Vasilets, L.A., LaTona, J., Schwarz, W. 1991. A negative slope in the current-voltage relationship of the Na+/K+ pump in Xenopus oocytes produced by reduction of external [K+]. J. Membrane Biol. 121:177–187Google Scholar
  19. Sachs, J.R. 1980. The order of release of sodium and addition of potassium in the sodium-potassium pump reaction mechanism. J. Physiol. 273:489–514Google Scholar
  20. Sachs, J.R., Welt, L.G. 1967. The concentration dependence of active potassium transport in the human red blood cell. J. Clin. Invest. 46:65–76Google Scholar
  21. Sagar, A., Rakowski, R.F. (1994). Access channel model for the voltage dependence of the forward-running Na+/K+ pump. J. Gen. Physiol. (in press)Google Scholar
  22. Schwartz, A., Nagano, K., Nakao, M., Lindenmeyer, G.E., Allen, J.C. 1971. The sodium- and potassium-activated adenosinetriphosphatase system. Methods Pharmacol. 1:361–388Google Scholar
  23. 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 face. J. Membrane Biol. 121:163–176Google Scholar

Copyright information

© Springer-Verlag New York Inc 1995

Authors and Affiliations

  • R. Bühler
    • 1
  • H. -J. Apell
    • 1
  1. 1.Department of BiologyUniversity of KonstanzKonstanzGermany

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