The Journal of Membrane Biology

, Volume 146, Issue 2, pp 211–224 | Cite as

Lumenal calcium modulates unitary conductance and gating of a plant vacuolar calcium release channel

  • E. Johannes
  • D. Sanders


The patch clamp technique has been used to investigate ion permeation and Ca2+-dependent gating of a voltage-sensitive Ca2+ release channel in the vacuolar membrane of sugar beet tap roots. Reversal potential measurements in bi-ionic conditions revealed a sequence for permeability ratios of Ca2+ ≈ Sr2+ ≈ Ba2+ > Mg2+ ≫ K+ which is inversely related to the size of the unitary conductances K+ ≫ Mg2+ ≈ Ba2+ > Sr2+ ≈ Ca2+, suggesting that ion movement is not independent. In the presence of Ca2+, the unitary K+ current is reduced in a concentration- and voltage-dependent manner by Ca2+ binding at a high affinity site (K0.5 = 0.29 mm at 0 mV) which is located 9% along the electric field of the membrane from the vacuolar side. Comparison of reversal potentials obtained under strictly bi-ionic conditions with those obtained in the presence of mixtures of the two ions indicates that the channel forms a multi-ion pore. Lumenal Ca2+ also has an effect on voltage-dependent channel gating. Stepwise increases of vacuolar Ca2+ from micromolar to millimolar concentrations resulted in a dramatic increase in channel openings over the physiological voltage range via a shift in threshold for channel activation to less negative membrane potentials. The steepness of the concentration dependence of channel activation by Ca2+ at −41 mV predicts that two Ca2+ ions need to bind to open the gate. The implications of the results for ion permeation and channel gating are discussed.

Key words

Calcium-permeable channel Permeation Selectivity Gating Calcium modulation Beta vulgaris 


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  1. Alexandre, J., Lassalles, J.P., Kado, R.T. 1990. Opening of Ca2+ channels in isolated red beet root vacuole membrane by inositol-1,4,5- trisphosphate. Nature 343:567–570Google Scholar
  2. Allen, G.J., Sanders, D. 1994a. Two voltage-gated, calcium release channels coreside in the vacuolar membrane of broad bean guard cells. Plant Cell 6:685–694Google Scholar
  3. Allen, G.J., Sanders, D. 1994b. Osmotic stress enhances the competence of Beta vulgaris vacuoles to respond to inositol 1,4,5- trisphosphate. Plant J. 6:687–695Google Scholar
  4. Barry, P.H., Lynch, J.W. 1991. Liquid junction potentials and small cell effects in patch-clamp analysis. J. Membrane Biol. 121:101–117Google Scholar
  5. Bertl, A., Blumwald, E., Coronado, R., Eisenberg, R., Findlay, G., Gradmann, D., Hille, B., Köhler, K., Kolb, H.A., MacRobbie, E., Meissner, G., Miller, C., Neher, E., Palade, P., Pantoja, O., Sanders, D., Schroeder, J., Slayman, C., Spanswick, R., Walker, A., Williams A. 1992. Electrical measurements on endomembranes. Science 258:873–874Google Scholar
  6. Bertl, A., Gradmann, D., Slayman, C.L. 1992. Calcium- and voltage-dependent ion channels in Saccharomyces cerevisiae. Phil. Trans. R. Soc. London B 338:63–72Google Scholar
  7. Bertl, A., Slayman, C.L. 1990. Cation-selective channels in the vacuolar membrane of Saccharomyces: Dependence on calcium, redox state, and voltage. Proc. Natl. Acad. Sci. USA 87:7824–7828.Google Scholar
  8. Brosnan, J.M., Sanders, D. 1990. Inositol trisphosphate-mediated Ca2+ release in beet microsomes is inhibited by heparin. FEBS Lett. 260:70–72Google Scholar
  9. Bush, D. 1993. Regulation of cytosolic calcium in plants. Plant Physiol. 103:7–13Google Scholar
  10. Cosgrove, D.J. & Hedrich, R. 1991. Stretch-activated chloride, potassium and calcium channels coexisting in plasma membranes of guard cells of Vicia faba L. Planta 186:143–153Google Scholar
  11. Ding, J.P., Pickard, B.G. 1993. Mechanosensory calcium-selective cation channels in epidermal cells. Plant J. 3:83–110Google Scholar
  12. Felle, H. 1988. Cytoplasmic free calcium in Riccia fluitans L. and Zea mays: Interaction of Ca2+ and pH? Planta 176:248–255Google Scholar
  13. Gelli, A., Blumwald, E. 1993. Calcium retrieval from vacuolar pools. Characterization of a vacuolar calcium channel. Plant Physiol. 102:1139–1146Google Scholar
  14. Gilroy, S., Fricker, M., Read, N.D., Trewavas, A.J. 1991. Role of calcium in signal transduction of Commelina guard cells. Plant Cell 3:333–344Google Scholar
  15. Green, W.N., Andersen, O.S. 1991. Surface charges and ion channel function. Annu. Rev. Physiol. 53:341–359Google Scholar
  16. Hamill, O.P., Marty, A., Neher, E., Sakmann, B., Sigworth, F.J. 1981. Improved patch clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluegers Arch. 391:85–100Google Scholar
  17. Hedrich, R., Neher, E. 1987. Cytoplasmic calcium regulates voltage-dependent ion channels in plant vacuoles. Nature 329:833–836Google Scholar
  18. Hille, B. 1992. Ionic channels of excitable membranes, 2nd edition. Sinauer Associates, Sunderland, MAGoogle Scholar
  19. Hille, B., Schwarz, W. 1978. Potassium channels as multi-ion single-file pores. J. Gen. Physiol. 72:409–442Google Scholar
  20. Johannes, E., Allen G.J., Sanders, D. 1994. Voltage-gated Ca2+ release channels in the vacuolar membranes from beet storage roots and guard cells. In: Membrane Transport in Plants and Fungi: Molecular Mechanisms and Control. SEB Symposia Vol. 48. M.R. Blatt, R.A. Leigh, and D. Sanders, editors, pp. 113–122. The Company of Biologists, Ltd., CambridgeGoogle Scholar
  21. Johannes, E., Brosnan, J.M., Sanders, D. 1991. Calcium channels and signal transduction in plant cells. Bioessays 13:331–336Google Scholar
  22. Johannes, E., Brosnan, J.M., Sanders, D. 1992a. Parallel pathways for intracellular Ca2+ release from the vacuole of higher plants. Plant J 2:97–102Google Scholar
  23. Johannes, E., Brosnan, J.M., Sanders, D. 1992b. Calcium channels in the vacuolar membrane of plants: multiple pathways for intracellular calcium mobilization. Phil. Trans. R. Soc. London B 338:105–112Google Scholar
  24. Klieber, H.-G., Gradmann, D. 1993. Enzyme kinetics of the prime K+ channel in the tonoplast of Chara: selectivity and inhibition. J. Membrane Biol. 132:253–265Google Scholar
  25. Läuger, P. 1976. Diffusion-limited ion flow through pores. Biochim. Biophys. Acta 455:493–509Google Scholar
  26. Laver, D.R. 1992. Divalent cation block and competition between divalent and monovalent cations in the large-conductance K+ channel from Chara australis. J. Gen. Physiol. 100:269–300Google Scholar
  27. Laver, D.R., Fairley, K.A., Walker, N.A. 1989. Ion permeation in a K+ channel in Chara australis: direct evidence for diffusion limitation of ion flow in a maxi-K+ channel. J. Membrane Biol. 108:153–164Google Scholar
  28. Laver, D.R., Fairley-Grenot, K.A. 1994. Surface potentials near the mouth of the large-conductance K+ channel from Chara australis: A new method of testing for diffusion-limited ion flow. J. Membrane Biol. 139:149–165Google Scholar
  29. Laver, D.R., Walker, N.A. 1991. Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops from Chara australis. J. Membrane Biol. 120:131–139Google Scholar
  30. Leigh, R.A., Wyn-Jones, R.G. 1984. A hypothesis relating critical potassium concentrations for growth to the distribution and functions of this ion in the plant cell New Phytol 97, 1–13Google Scholar
  31. Lewis, C.A., Stevens, C.F. 1979. Mechanism of ion permeation through channels in a postsynaptic membrane. In: Membrane Transport Processes, Vol. 3. C.F. Stevens, and R.W. Tsien, editors, pp. 133–151. Raven Press, New YorkGoogle Scholar
  32. Maathuis, F.J.M., Sanders, D. 1993. Energization of potassium uptake in Arabidopsis thaliana. Planta 191:302–307Google Scholar
  33. Neher, E. 1992. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207:123–131Google Scholar
  34. Pantoja, O., Gelli, A., Blumwald, E. 1992. Voltage-dependent calcium channels in plant vacuoles. Science 255:1567–1570Google Scholar
  35. Piñeros, M., Tester, M. 1995. Characterization of a voltage-dependent Ca2+-selective channel from wheat roots. Planta, 195:478–488Google Scholar
  36. Ping, Z., Yabe, I., Muto, S. 1992a. Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana. Biochim. Biophys. Acta 112:287–290Google Scholar
  37. Ping, Z., Yabe, I., Muto, S. 1992b Identification of K+, Cl, and Ca2+ channels in the vacuolar membrane of tobacco cell suspension cultures. Protoplasma 171:7–18Google Scholar
  38. Plant, P.J., Gelli, A., Blumwald, E. 1994. Vacuolar chloride regulation of an anion-selective tonoplast channel. J. Membrane Biol. 140:1–12Google Scholar
  39. Rea, P.A., Sanders, D. 1987. Tonoplast energization: Two H+ pumps, one membrane. Physiol. Plant. 71:131–141Google Scholar
  40. Rosenberg, R.L., Chen, X.-H. 1991. Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels. J. Gen. Physiol. 97:1207–1225Google Scholar
  41. Schroeder, J.I., Thuleau, P. 1991. Ca2+ channels in higher plant cells. Plant Cell 3:555–559Google Scholar
  42. Skoog, D.A., West, D.M. 1982. Fundamentals of analytical chemistry. Saunders College Publishing, PhiladelphiaGoogle Scholar
  43. Tsien, R.W., Hess, P., McCleskey, E.W., Rosenberg, R.L. 1987. Calcium channels: Mechanisms of selectivity, permeation, and block. Ann. Rev. Biophys. Biophys. Chem. 16:265–290Google Scholar
  44. Thuleau, P., Ward, J.M., Ranjeva, R., Schroeder, J.I. 1994. Voltage-dependent calcium-permeable channels in the plasma membrane of a higher plant cell. EMBO J. 13:2970–2975Google Scholar
  45. Ward, J.M., Schroeder, J.I. 1994. Calcium-activated K+ channels and calcium-induced calcium release by slow vacuolar ion channels in guard cell vacuoles implicated in the control of stomatal closure. Plant Cell 6:669–683Google Scholar
  46. Woodhull, A.M. 1973. Ionic blockage of sodium channels in nerve. J. Gen. Physiol. 61:687–708CrossRefPubMedGoogle Scholar
  47. Yang, J., Ellinor, P.T., Sather, W.A., Zhang, J.F., Tsien, R.W. 1993. Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. Nature 366:158–161Google Scholar

Copyright information

© Springer-Verlag New York Inc 1995

Authors and Affiliations

  • E. Johannes
    • 1
  • D. Sanders
    • 1
  1. 1.The Plant Laboratory, Biology DepartmentUniversity of YorkYorkUK

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