The Journal of Membrane Biology

, Volume 95, Issue 2, pp 105–112 | Cite as

Reconstitution in phospholipid vesicles of calcium-activated potassium channel from outer renal medulla

  • Dan A. Klaerke
  • Steven J. D. Karlish
  • Peter L. Jørgensen
Articles

Summary

A barium-sensitive Ca-activated K+ channel in the luminal membrane of the tubule cells in thick ascending limb of Henle's loop is required for maintenance of the lumen positive transepithelial potential and may be important for regulation of NaCl reabsorption. In this paper we examine if the K+ channel can be solubilized and reconstituted into phospholipid vesicles with preservation of its native properties. The K+ channel in luminal plasma membrane vesicles can be quantitatively solubilized in CHAPS at a detergent/protein ratio of 3. For reconstitution, detergent is removed by passage over a column of Sephadex G 50 (coarse). K+-channel activity is assayed by measurement of86Rb+ uptake against a large opposing K+ gradient. The reconstituted K+ channel is activated by Ca2+ in the physiological range of concentration (K1/2∼2×10−7m at pH 7.2) as found for the K+ channel in native plasma membrane vesicles and shows the same sensitivity to inhibitors (Ba2+, trifluoperazine, calmidazolium, quinidine) and to protons. Reconstitution of the K+ channel into phospholipid vesicles with full preservation of its native properties is an essential step towards isolation and purification of the K+-channel protein.

Titration with Ca2+ shows that most of the active K+ channels in reconstituted vesicles have their cytoplasmic aspect facing outward in contrast to the orientation in plasma membrane vesicles, which requires also addition of Ca2+ ionophore in order to observe Ca2+ stimulation. The reconstituted K+ channel is highly sensitive to tryptic digestion. Brief digestion leads to activation of the K+ channel in absence of Ca2+, to the level of activity seen with saturating concentrations of Ca2+. This tryptic split is located in a cytoplasmic aspect of the K+ channel that appears to be involved in opening and closing the K+ channel in response to Ca2+ binding.

Key Words

Ca-activated K+ channel solubilization reconstitution thick ascending limb of Henle's loop calmodulin inhibitors trypsin pH 

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References

  1. 1.
    Anderson, K.W., Coll, R.J., Murphy, A.J. 1984. Inhibition of skeletal muscle sarcoplasmic reticulum CaATPase activity by calmidazolium.J. Biol. Chem. 259:11487–11490Google Scholar
  2. 2.
    Biagi, B., Sohtell, M., Giebisch, G. 1981. Intracellular potassium activity in the rabbit proximal straight tubule.Am. J. Physiol. 241:F677-F686Google Scholar
  3. 3.
    Burnham, C., Braw, R., Karlish, S.J.D. 1986. A Ca-dependent K channel in “luminal” membranes from the renal outer medulla.J. Membrane Biol. 93:177–186Google Scholar
  4. 4.
    Burnham, C., Karlish, S.J.D., Jøgensen, P.L. 1985. Identification and reconstitution of a Na+/K+/Cl cotransporter and K+ channel from luminal membranes of renal red outer medulla.Biochim. Biophys. Acta 821:461–469Google Scholar
  5. 5.
    Christensen, O., Zeuthen, T. 1987. Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential.Pfleuger's Arch. (in press) Google Scholar
  6. 6.
    Cook, D.L., Ikeuchi, M., Fujimoto, W.Y. 1984. Lowering of pHi inhibits Ca2+-activated K+ channels in pancreatic B-cells.Nature (London) 311:269–273Google Scholar
  7. 7.
    Dallner, G. 1978. Isolation of microsomal subfractions by use of density gradients.Methods Enzymol. 52:71–83Google Scholar
  8. 8.
    Eaton, D.C., Malcolm, B.S. 1980. Effects of barium on the potassium conductance of squid axon.J. Gen. Physiol. 75:727–750Google Scholar
  9. 9.
    Findlay, I. 1984. A patch-clamp study of potassium channels and whole-cell currents in acinar cells of the mouse lacrimal gland.J. Physiol. (London) 350:179–195Google Scholar
  10. 10.
    Garty, H., Rudy, B., Karlish, S.J.D. 1983. A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles.J. Biol. Chem. 258:13094–13099Google Scholar
  11. 11.
    Gietzen, K., Sadorf, I., Bader, H. 1982. A model for the regulation of the calmodulin-dependent enzymes erythrocyte Ca2+-transport ATPase and brain phosphodiesterase by activators and inhibitors.Biochem. J. 207:541–548Google Scholar
  12. 12.
    Gietzen, K., Wütrich, A., Bader, H. 1981. R 24571: A new powerful inhibitor of red blood cell Ca2+-transport ATPase and of calmodulin-regulated functions.Biochem. Biophys. Res. Commun. 101:418–425Google Scholar
  13. 13.
    Good, N.E., Izawa, S. 1972. Hydrogen ion buffers.Methods Enzymol. 24:53–68Google Scholar
  14. 14.
    Greger, R., Schlatter, E. 1983. Properties of the basolateral membrane of the cortical thick ascending limb of Henle's loop of rabbit kidney.Pfluegers Arch. 396:325–334Google Scholar
  15. 15.
    Helenius, A., Simons, K. 1975. Solubilization of membranes by detergents.Biochim. Biophys. Acta 415:29–79Google Scholar
  16. 16.
    Herbert, S.C., Friedman, P.A., Andreoli, T.E. 1984. Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways.J. Membrane Biol. 80:201–219Google Scholar
  17. 17.
    Hill, T.L., Inesi, G. 1982. Equilibrium cooperative binding of calcium and protons by sarcoplasmic reticulum ATPase.Proc. Natl. Acad. Sci. USA 79:3978–3982Google Scholar
  18. 18.
    Hjelmeland, L.M. 1980. A nondenaturing zwitterionic detergent for membrane biochemistry: Design and synthesis.Proc. Natl. Acad. Sci. USA 77:6368–6370Google Scholar
  19. 19.
    Ho, M.-M., Scales, D.J., Inesi, G. 1983. The effect of trifluoroperazine on the sarcoplasmic reticulum membrane.Biochim. Biophys. Acta 730:64–70Google Scholar
  20. 20.
    Iwatsuki, N., Petersen, O.H. 1985. Inhibition of Ca2+-activated K+-channels in pig pancreatic acinar cells by Ba2+, Ca2+, quinine and quinidine.Nature (London) 819:249–257Google Scholar
  21. 21.
    Jørgensen, P.L. 1974. 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
  22. 22.
    Jørgensen, P.L. 1975. Purification and characterization of (Na+, K+)-ATPase: V. Conformational changes in the enzyme. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.Biochim. Biophys. Acta 401:399–415Google Scholar
  23. 23.
    Kasahara, M., Hinkle, P.C. 1977. Reconstitution and purification of thed-glucose transporter from human erythrocytes.J. Biol. Chem. 252:7384–7390Google Scholar
  24. 24.
    Latorre, R., Miller, C. 1983. Conduction and selectivity in potassium channels.J. Membrane Biol. 71:11–30Google Scholar
  25. 25.
    Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275Google Scholar
  26. 26.
    Moody, W.J., Hagiwara, S. 1982. Block of inward retification by intracellular H+ in immature oocytes of the starfishMediaster aequalis.J. Gen. Physiol. 79:115–130Google Scholar
  27. 27.
    Pape, L., Kristensen, B.L. 1984. A calmodulin activated Ca2+-dependent K+ channel in human erythrocyte membrane inside-out vesicles.Biochim. Biophys. Acta 770:1–6Google Scholar
  28. 28.
    Penefsky, H.S. 1977. Reversible binding of Pi by beef heart mitochondrial adenosine triphosphatase.J. Biol. Chem. 252:2891–2899Google Scholar
  29. 29.
    Pershadsingh, H.A., Gale, R.D., Delfert, D.M., McDonald, J.M. 1986. A calmodulin dependent Ca2+-activated K_ channel in the adipocyte plasma membrane.Biochem. Biophys. Res. Commun. 135:934–941Google Scholar
  30. 30.
    Pershadsingh, H.A., McDonald, J.M. 1980. A highly affinity calcium-stimulated magnesium-dependent triphosphatase in rat adipocyte plasma membrane.J. Biol. Chem. 255:4087–4093Google Scholar
  31. 31.
    Petersen, O.H., Maruyama, Y. 1984. Calcium-acivated potassium channels and their role in secretion.Nature (Nature) 307:693–696Google Scholar
  32. 32.
    Reichstein, E., Rothstein, A. 1981. Effects of quinine on Ca2+-induced K+ efflux from human red blood cells.J. Membrane Biol. 59:57–63Google Scholar
  33. 33.
    Schlatter, E., Greger, R. 1985. cAMP increases in the basolateral Cl-conductance in the isolated perfused medullary thick ascending limb of Henle's loop of the mouse.Pfluegers Arch. 405:367–376Google Scholar
  34. 34.
    Simonsen, L.O., Gomme, G., Lew, V.L. 1982. Uniform ionophore A23187 distribution and cytoplasmic calcium buffering in intact human red cells.Biochim. Biophys. Acta 692:431–440Google Scholar
  35. 35.
    Stampe, P., Vestergaard-Bogind, B. 1985. The Ca2+-sensitive K+-conductance of the human red cell membrane is strongly dependent on cellular pH.Biochim. Biophys. Acta 815:313–321Google Scholar
  36. 36.
    Wanke, E., Carbone, E., Testa, P.L. 1979. K+ conductance modified by a titratable group accessible to protons from the intracellular side of the squid axon membrane.Biophys. J. 26:319–324Google Scholar
  37. 37.
    Wood, P.G., Mueller, H. 1984. Modification of the cation selectivity filter and the calcium receptor of the Ca-stimulated K channel in resealed ghosts of human red blood cells by low levels of incorporated trypsin.Eur. J. Biochem. 141:91–95Google Scholar
  38. 38.
    Wood, P.G., Mueller, H. 1984. The effects of terbium(III) on the Ca-activated K channel found in the resealed human erythrocyte membrane.Eur. J. Biochem. 146:65–69Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1987

Authors and Affiliations

  • Dan A. Klaerke
    • 1
  • Steven J. D. Karlish
    • 2
  • Peter L. Jørgensen
    • 1
  1. 1.Institute of PhysiologyAarhus UniversityAarhus CDenmark
  2. 2.Department of BiochemistryWeizmann Institute of ScienceRehovotIsrael

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