Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 May;74(5):2285–2298. doi: 10.1016/S0006-3495(98)77938-X

Fast transient currents in Na,K-ATPase induced by ATP concentration jumps from the P3-[1-(3',5'-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP.

V S Sokolov 1, H J Apell 1, J E Corrie 1, D R Trentham 1
PMCID: PMC1299572  PMID: 9591656

Abstract

Electrogenic ion transport by Na,K-ATPase was investigated by analysis of transient currents in a model system of protein-containing membrane fragments adsorbed to planar lipid bilayers. Sodium transport was triggered by ATP concentration jumps in which ATP was released from an inactive precursor by an intense near-UV light flash. The method has been used previously with the P3-1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), from which the relatively slow rate of ATP release limits analysis of processes in the pump mechanism controlled by rate constants greater than 100 s(-1) at physiological pH. Here Na,K-ATPase was reinvestigated using the P3-[1-(3,5-dimethoxyphenyl)-2-phenyl-2-oxo]ethyl ester of ATP (DMB-caged ATP), which has an ATP release rate of >10(5) s(-1). Under otherwise identical conditions, photorelease of ATP from DMB-caged ATP showed faster kinetics of the transient current compared to that from NPE-caged ATP. With DMB-caged ATP, transient currents had rate profiles that were relatively insensitive to pH and the concentration of caged compound. Rate constants of ATP binding and of the E1 to E2 conformational change were compatible with earlier studies. Rate constants of enzyme phosphorylation and ADP-dependent dephosphorylation were 600 s(-1) and 1.5 x 10(6) M(-1) s(-1), respectively, at pH 7.2 and 22 degrees C.

Full Text

The Full Text of this article is available as a PDF (275.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apell H. J., Borlinghaus R., Läuger P. Fast charge translocations associated with partial reactions of the Na,K-pump: II. Microscopic analysis of transient currents. J Membr Biol. 1987;97(3):179–191. doi: 10.1007/BF01869221. [DOI] [PubMed] [Google Scholar]
  2. Apell H. J., Marcus M. M. (Na+ + K+)-ATPase in artificial lipid vesicles: influence of the concentration of mono- and divalent cations on the pumping rate. Biochim Biophys Acta. 1986 Nov 17;862(2):254–264. doi: 10.1016/0005-2736(86)90226-9. [DOI] [PubMed] [Google Scholar]
  3. Apell H. J., Nelson M. T., Marcus M. M., Läuger P. Effects of the ATP, ADP and inorganic phosphate on the transport rate of the Na+,K+-pump. Biochim Biophys Acta. 1986 May 9;857(1):105–115. doi: 10.1016/0005-2736(86)90103-3. [DOI] [PubMed] [Google Scholar]
  4. Apell H. J., Roudna M., Corrie J. E., Trentham D. R. Kinetics of the phosphorylation of Na,K-ATPase by inorganic phosphate detected by a fluorescence method. Biochemistry. 1996 Aug 20;35(33):10922–10930. doi: 10.1021/bi960238t. [DOI] [PubMed] [Google Scholar]
  5. Borlinghaus R., Apell H. J. Current transients generated by the Na+/K+-ATPase after an ATP concentration jump: dependence on sodium and ATP concentration. Biochim Biophys Acta. 1988 Apr 7;939(2):197–206. doi: 10.1016/0005-2736(88)90063-6. [DOI] [PubMed] [Google Scholar]
  6. Borlinghaus R., Apell H. J., Läuger P. Fast charge translocations associated with partial reactions of the Na,K-pump: I. Current and voltage transients after photochemical release of ATP. J Membr Biol. 1987;97(3):161–178. doi: 10.1007/BF01869220. [DOI] [PubMed] [Google Scholar]
  7. Bühler R., Stürmer W., Apell H. J., Läuger P. Charge translocation by the Na,K-pump: I. Kinetics of local field changes studied by time-resolved fluorescence measurements. J Membr Biol. 1991 Apr;121(2):141–161. doi: 10.1007/BF01870529. [DOI] [PubMed] [Google Scholar]
  8. Fendler K., Grell E., Bamberg E. Kinetics of pump currents generated by the Na+,K+-ATPase. FEBS Lett. 1987 Nov 16;224(1):83–88. doi: 10.1016/0014-5793(87)80427-1. [DOI] [PubMed] [Google Scholar]
  9. Fendler K., Grell E., Haubs M., Bamberg E. Pump currents generated by the purified Na+K+-ATPase from kidney on black lipid membranes. EMBO J. 1985 Dec 1;4(12):3079–3085. doi: 10.1002/j.1460-2075.1985.tb04048.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fendler K., Jaruschewski S., Hobbs A., Albers W., Froehlich J. P. Pre-steady-state charge translocation in NaK-ATPase from eel electric organ. J Gen Physiol. 1993 Oct;102(4):631–666. doi: 10.1085/jgp.102.4.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Forbush B., 3rd An apparatus for rapid kinetic analysis of isotopic efflux from membrane vesicles and of ligand dissociation from membrane proteins. Anal Biochem. 1984 Aug 1;140(2):495–505. doi: 10.1016/0003-2697(84)90200-8. [DOI] [PubMed] [Google Scholar]
  12. Forbush B., 3rd Na+ movement in a single turnover of the Na pump. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5310–5314. doi: 10.1073/pnas.81.17.5310. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friedrich T., Bamberg E., Nagel G. Na+,K(+)-ATPase pump currents in giant excised patches activated by an ATP concentration jump. Biophys J. 1996 Nov;71(5):2486–2500. doi: 10.1016/S0006-3495(96)79442-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Friedrich T., Nagel G. Comparison of Na+/K(+)-ATPase pump currents activated by ATP concentration or voltage jumps. Biophys J. 1997 Jul;73(1):186–194. doi: 10.1016/S0006-3495(97)78059-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gadsby D. C., Nakao M., Bahinski A. Voltage-induced Na/K pump charge movements in dialyzed heart cells. Soc Gen Physiol Ser. 1991;46:355–371. [PubMed] [Google Scholar]
  16. Gadsby D. C., Rakowski R. F., De Weer P. Extracellular access to the Na,K pump: pathway similar to ion channel. Science. 1993 Apr 2;260(5104):100–103. doi: 10.1126/science.7682009. [DOI] [PubMed] [Google Scholar]
  17. Heyse S., Wuddel I., Apell H. J., Stürmer W. Partial reactions of the Na,K-ATPase: determination of rate constants. J Gen Physiol. 1994 Aug;104(2):197–240. doi: 10.1085/jgp.104.2.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hilgemann D. W. Channel-like function of the Na,K pump probed at microsecond resolution in giant membrane patches. Science. 1994 Mar 11;263(5152):1429–1432. doi: 10.1126/science.8128223. [DOI] [PubMed] [Google Scholar]
  19. Jorgensen P. L. Isolation of (Na+ plus K+)-ATPase. Methods Enzymol. 1974;32:277–290. [PubMed] [Google Scholar]
  20. Kane D. J., Fendler K., Grell E., Bamberg E., Taniguchi K., Froehlich J. P., Clarke R. J. Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase. Biochemistry. 1997 Oct 28;36(43):13406–13420. doi: 10.1021/bi970598w. [DOI] [PubMed] [Google Scholar]
  21. Keillor J. W., Jencks W. P. Phosphorylation of the sodium--potassium adenosinetriphosphatase proceeds through a rate-limiting conformational change followed by rapid phosphoryl transfer. Biochemistry. 1996 Feb 27;35(8):2750–2753. doi: 10.1021/bi951370g. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Lu C. C., Kabakov A., Markin V. S., Mager S., Frazier G. A., Hilgemann D. W. Membrane transport mechanisms probed by capacitance measurements with megahertz voltage clamp. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):11220–11224. doi: 10.1073/pnas.92.24.11220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McCray J. A., Trentham D. R. Properties and uses of photoreactive caged compounds. Annu Rev Biophys Biophys Chem. 1989;18:239–270. doi: 10.1146/annurev.bb.18.060189.001323. [DOI] [PubMed] [Google Scholar]
  25. Märdh S., Post R. L. Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme. J Biol Chem. 1977 Jan 25;252(2):633–638. [PubMed] [Google Scholar]
  26. Nagel G., Fendler K., Grell E., Bamberg E. Na+ currents generated by the purified (Na+ + K+)-ATPase on planar lipid membranes. Biochim Biophys Acta. 1987 Jul 23;901(2):239–249. doi: 10.1016/0005-2736(87)90120-9. [DOI] [PubMed] [Google Scholar]
  27. Nakao M., Gadsby D. C. Voltage dependence of Na translocation by the Na/K pump. Nature. 1986 Oct 16;323(6089):628–630. doi: 10.1038/323628a0. [DOI] [PubMed] [Google Scholar]
  28. Rakowski R. F., Vasilets L. A., LaTona J., Schwarz W. A negative slope in the current-voltage relationship of the Na+/K+ pump in Xenopus oocytes produced by reduction of external [K+]. J Membr Biol. 1991 Apr;121(2):177–187. doi: 10.1007/BF01870531. [DOI] [PubMed] [Google Scholar]
  29. Sagar A., Rakowski R. F. Access channel model for the voltage dependence of the forward-running Na+/K+ pump. J Gen Physiol. 1994 May;103(5):869–893. doi: 10.1085/jgp.103.5.869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schulz S., Apell H. J. Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump. Eur Biophys J. 1995;23(6):413–421. doi: 10.1007/BF00196828. [DOI] [PubMed] [Google Scholar]
  31. Thirlwell H., Corrie J. E., Reid G. P., Trentham D. R., Ferenczi M. A. Kinetics of relaxation from rigor of permeabilized fast-twitch skeletal fibers from the rabbit using a novel caged ATP and apyrase. Biophys J. 1994 Dec;67(6):2436–2447. doi: 10.1016/S0006-3495(94)80730-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Thirlwell H., Sleep J. A., Ferenczi M. A. Inhibition of unloaded shortening velocity in permeabilized muscle fibres by caged ATP compounds. J Muscle Res Cell Motil. 1995 Apr;16(2):131–137. doi: 10.1007/BF00122531. [DOI] [PubMed] [Google Scholar]
  33. Vasilets L. A., Omay H. S., Ohta T., Noguchi S., Kawamura M., Schwarz W. Stimulation of the Na+/K+ pump by external [K+] is regulated by voltage-dependent gating. J Biol Chem. 1991 Sep 5;266(25):16285–16288. [PubMed] [Google Scholar]
  34. Wuddel I., Apell H. J. Electrogenicity of the sodium transport pathway in the Na,K-ATPase probed by charge-pulse experiments. Biophys J. 1995 Sep;69(3):909–921. doi: 10.1016/S0006-3495(95)79965-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES