Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1992 Jan 1;99(1):41–62. doi: 10.1085/jgp.99.1.41

Calmodulin activation of the Ca2+ pump revealed by fluorescent chelator dyes in human red blood cell ghosts

PMCID: PMC2216596  PMID: 1371307

Abstract

Ca2+ transport in red blood cell ghosts was monitored with fura2 or quin2 incorporated as the free acid during resealing. This is the first report of active transport monitored by the fluorescent intensity of the chelator dyes fura2 (5-50 microM) or quin2 (250 microM) in hemoglobin-depleted ghosts. Since there are no intracellular compartments in ghosts and the intracellular concentrations of all assay chelator substances including calmodulin (CaM), the dyes, and ATP could be set, the intracellular concentrations of free and total Ca [( Cafree]i and [Catotal]i) could be calculated during the transport. Ghosts prepared with or without CaM rapidly extruded Ca2+ to a steady- state concentration of 60-100 nM. A 10(4)-fold gradient for Ca2+ was routinely produced in medium containing 1 mM Ca2+. During active Ca2+ extrusion, d[Cafree]i/dt was a second order function of [Cafree]i and was independent of the dye concentration, whereas d[Catotal]i/dt increased as a first order function of both the [Cafree]i and the concentration of the Ca:dye complex. CaM (5 microM) increased d[Catotal]i/dt by 400% at 1 microM [Cafree]i, while d[Cafree]i/dt increased by only 25%. From a series of experiments we conclude that chelated forms of Ca2+ serve as substrates for the pump under permissive control of the [Cafree]i, and this dual effect may explain cooperativity. Free Ca2+ is extruded, and probably also Ca2+ bound to CaM or other chelators, while CaM and the chelators are retained in the cell.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Blinks J. R., Wier W. G., Hess P., Prendergast F. G. Measurement of Ca2+ concentrations in living cells. Prog Biophys Mol Biol. 1982;40(1-2):1–114. doi: 10.1016/0079-6107(82)90011-6. [DOI] [PubMed] [Google Scholar]
  2. Bond G. H., Clough D. L. A soluble protein activator of (Mg2+ plus Ca2+)-dependent ATPase in human red cell membranes. Biochim Biophys Acta. 1973 Nov 16;323(4):592–599. doi: 10.1016/0005-2736(73)90167-3. [DOI] [PubMed] [Google Scholar]
  3. Cantley L. C., Jr, Resh M. D., Guidotti G. Vanadate inhibits the red cell (Na+, K+) ATPase from the cytoplasmic side. Nature. 1978 Apr 6;272(5653):552–554. doi: 10.1038/272552a0. [DOI] [PubMed] [Google Scholar]
  4. Carafoli E. Calcium pump of the plasma membrane. Physiol Rev. 1991 Jan;71(1):129–153. doi: 10.1152/physrev.1991.71.1.129. [DOI] [PubMed] [Google Scholar]
  5. David-Dufilho M., Montenay-Garestier T., Devynck M. A. Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2. Cell Calcium. 1988 Aug;9(4):167–179. doi: 10.1016/0143-4160(88)90021-8. [DOI] [PubMed] [Google Scholar]
  6. Enyedi A., Flura M., Sarkadi B., Gardos G., Carafoli E. The maximal velocity and the calcium affinity of the red cell calcium pump may be regulated independently. J Biol Chem. 1987 May 5;262(13):6425–6430. [PubMed] [Google Scholar]
  7. Farrance M. L., Vincenzi F. F. Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. II. Dependence on calcium and a cytoplasmic activator. Biochim Biophys Acta. 1977 Nov 15;471(1):59–66. doi: 10.1016/0005-2736(77)90393-5. [DOI] [PubMed] [Google Scholar]
  8. Foder B., Scharff O. Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations. Biochim Biophys Acta. 1981 Dec 7;649(2):367–376. doi: 10.1016/0005-2736(81)90426-0. [DOI] [PubMed] [Google Scholar]
  9. Glynn I. M., Hoffman J. F. Nucleotide requirements for sodium-sodium exchange catalysed by the sodium pump in human red cells. J Physiol. 1971 Oct;218(1):239–256. doi: 10.1113/jphysiol.1971.sp009612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  11. Highsmith S., Bloebaum P., Snowdowne K. W. Sarcoplasmic reticulum interacts with the Ca(2+) indicator precursor fura-2-am. Biochem Biophys Res Commun. 1986 Aug 14;138(3):1153–1162. doi: 10.1016/s0006-291x(86)80403-x. [DOI] [PubMed] [Google Scholar]
  12. Jackson A. P., Timmerman M. P., Bagshaw C. R., Ashley C. C. The kinetics of calcium binding to fura-2 and indo-1. FEBS Lett. 1987 May 25;216(1):35–39. doi: 10.1016/0014-5793(87)80752-4. [DOI] [PubMed] [Google Scholar]
  13. Klee C. B., Crouch T. H., Richman P. G. Calmodulin. Annu Rev Biochem. 1980;49:489–515. doi: 10.1146/annurev.bi.49.070180.002421. [DOI] [PubMed] [Google Scholar]
  14. Knauf P. A., Fuhrmann G. F., Rothstein S., Rothstein A. The relationship between anion exchange and net anion flow across the human red blood cell membrane. J Gen Physiol. 1977 Mar;69(3):363–386. doi: 10.1085/jgp.69.3.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kotagal N., Colca J. R., McDaniel M. L. Activation of an islet cell plasma membrane (Ca2+ + Mg2+)-ATPase by calmodulin and Ca-EGTA. J Biol Chem. 1983 Apr 25;258(8):4808–4813. [PubMed] [Google Scholar]
  16. Kratje R. B., Garrahan P. J., Rega A. F. The effects of alkali metal ions on active Ca2+ transport in reconstituted ghosts from human red cells. Biochim Biophys Acta. 1983 May 26;731(1):40–46. doi: 10.1016/0005-2736(83)90395-4. [DOI] [PubMed] [Google Scholar]
  17. Larsen F. L., Hinds T. R., Vincenzi F. F. On the red blood cell Ca2+-pump: an estimate of stoichiometry. J Membr Biol. 1978 Jul 18;41(4):361–376. doi: 10.1007/BF01872000. [DOI] [PubMed] [Google Scholar]
  18. Lew V. L., Muallem S., Seymour C. A. Properties of the Ca2+-activated K+ channel in one-step inside-out vesicles from human red cell membranes. Nature. 1982 Apr 22;296(5859):742–744. doi: 10.1038/296742a0. [DOI] [PubMed] [Google Scholar]
  19. Lew V. L., Tsien R. Y., Miner C., Bookchin R. M. Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator. Nature. 1982 Jul 29;298(5873):478–481. doi: 10.1038/298478a0. [DOI] [PubMed] [Google Scholar]
  20. Mualem S., Karlish S. J. Is the red cell calcium pump regulated by ATP? Nature. 1979 Jan 18;277(5693):238–240. doi: 10.1038/277238a0. [DOI] [PubMed] [Google Scholar]
  21. Muallem S., Karlish S. J. Regulation of the Ca2+-pump by calmodulin in intact cells. Biochim Biophys Acta. 1982 May 7;687(2):329–332. doi: 10.1016/0005-2736(82)90563-6. [DOI] [PubMed] [Google Scholar]
  22. Murphy E., Levy L., Berkowitz L. R., Orringer E. P., Gabel S. A., London R. E. Nuclear magnetic resonance measurement of cytosolic free calcium levels in human red blood cells. Am J Physiol. 1986 Oct;251(4 Pt 1):C496–C504. doi: 10.1152/ajpcell.1986.251.4.C496. [DOI] [PubMed] [Google Scholar]
  23. Niggli V., Adunyah E. S., Penniston J. T., Carafoli E. Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids. J Biol Chem. 1981 Jan 10;256(1):395–401. [PubMed] [Google Scholar]
  24. Plishker G. A., White P. H., Cadman E. D. Involvement of a cytoplasmic protein in calcium-dependent potassium efflux in red blood cells. Am J Physiol. 1986 Oct;251(4 Pt 1):C535–C540. doi: 10.1152/ajpcell.1986.251.4.C535. [DOI] [PubMed] [Google Scholar]
  25. Quast U., Labhardt A. M., Doyle V. M. Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions. Biochem Biophys Res Commun. 1984 Sep 17;123(2):604–611. doi: 10.1016/0006-291x(84)90272-9. [DOI] [PubMed] [Google Scholar]
  26. Quist E. E., Roufogalis B. D. Determination of the stoichiometry of the calcium pump in human erythrocytes using lanthanum as a selective inhibitor. FEBS Lett. 1975 Feb 1;50(2):135–139. doi: 10.1016/0014-5793(75)80473-x. [DOI] [PubMed] [Google Scholar]
  27. Rasmussen H. The cycling of calcium as an intracellular messenger. Sci Am. 1989 Oct;261(4):66–73. doi: 10.1038/scientificamerican1089-66. [DOI] [PubMed] [Google Scholar]
  28. Rossi J. P., Garrahan P. J., Rega A. F. Vanadate inhibition of active Ca2+ transport across human red cell membranes. Biochim Biophys Acta. 1981 Nov 6;648(2):145–150. doi: 10.1016/0005-2736(81)90029-8. [DOI] [PubMed] [Google Scholar]
  29. Sarkadi B., Enyedi A., Gárdos G. Conformational changes of the in situ red cell membrane calcium pump affect its proteolysis. Biochim Biophys Acta. 1987 May 12;899(1):129–133. doi: 10.1016/0005-2736(87)90247-1. [DOI] [PubMed] [Google Scholar]
  30. Sarkadi B., Schubert A., Gárdos G. Effects of calcium-EGTA buffers on active calcium transport in inside-out red cell membrane vesicles. Experientia. 1979 Aug 15;35(8):1045–1047. doi: 10.1007/BF01949932. [DOI] [PubMed] [Google Scholar]
  31. Scanlon M., Williams D. A., Fay F. S. A Ca2+-insensitive form of fura-2 associated with polymorphonuclear leukocytes. Assessment and accurate Ca2+ measurement. J Biol Chem. 1987 May 5;262(13):6308–6312. [PubMed] [Google Scholar]
  32. Scharff O., Foder B. Rate constants for calmodulin binding to Ca2+-ATPase in erythrocyte membranes. Biochim Biophys Acta. 1982 Sep 24;691(1):133–143. doi: 10.1016/0005-2736(82)90222-x. [DOI] [PubMed] [Google Scholar]
  33. Scharff O., Foder B., Skibsted U. Hysteretic activation of the Ca2+ pump revealed by calcium transients in human red cells. Biochim Biophys Acta. 1983 May 5;730(2):295–305. doi: 10.1016/0005-2736(83)90346-2. [DOI] [PubMed] [Google Scholar]
  34. Schatzmann H. J. Dependence on calcium concentration and stoichiometry of the calcium pump in human red cells. J Physiol. 1973 Dec;235(2):551–569. doi: 10.1113/jphysiol.1973.sp010403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Taverna R. D., Hanahan D. J. Modulation of human erythrocyte Ca2+/Mg2+ ATPase activity by phospholipase A2 and proteases. A comparison with calmodulin. Biochem Biophys Res Commun. 1980 May 30;94(2):652–659. doi: 10.1016/0006-291x(80)91282-6. [DOI] [PubMed] [Google Scholar]
  36. Tiffert T., Garcia-Sancho J., Lew V. L. Irreversible ATP depletion caused by low concentrations of formaldehyde and of calcium-chelator esters in intact human red cells. Biochim Biophys Acta. 1984 Jun 13;773(1):143–156. doi: 10.1016/0005-2736(84)90559-5. [DOI] [PubMed] [Google Scholar]
  37. Tsien R. Y., Pozzan T., Rink T. J. Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator. J Cell Biol. 1982 Aug;94(2):325–334. doi: 10.1083/jcb.94.2.325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Uto A., Arai H., Ogawa Y. Reassessment of Fura-2 and the ratio method for determination of intracellular Ca2+ concentrations. Cell Calcium. 1991 Jan;12(1):29–37. doi: 10.1016/0143-4160(91)90082-p. [DOI] [PubMed] [Google Scholar]
  39. Varecka L., Carafoli E. Vanadate-induced movements of Ca2+ and K+ in human red blood cells. J Biol Chem. 1982 Jul 10;257(13):7414–7421. [PubMed] [Google Scholar]
  40. Villalobo A., Brown L., Roufogalis B. D. Kinetic properties of the purified Ca2+-translocating ATPase from human erythrocyte plasma membrane. Biochim Biophys Acta. 1986 Jan 16;854(1):9–20. doi: 10.1016/0005-2736(86)90059-3. [DOI] [PubMed] [Google Scholar]
  41. Vincenzi F. F., Hinds T. R., Raess B. U. Calmodulin and the plasma membrane calcium pump. Ann N Y Acad Sci. 1980;356:232–244. doi: 10.1111/j.1749-6632.1980.tb29614.x. [DOI] [PubMed] [Google Scholar]
  42. Yingst D. R., Hoffman J. F. Changes of intracellular Ca++ as measured by arsenazo III in relation to the K permeability of human erythrocyte ghosts. Biophys J. 1978 Sep;23(3):463–471. doi: 10.1016/S0006-3495(78)85462-9. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES