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Planta

, Volume 178, Issue 4, pp 509–523 | Cite as

Mechanisms of fusicoccin action: kinetic modification and inactivation of K+ channels in guard cells

  • Michael R. Blatt
  • Gill M. Clint
Article

Abstract

Fusicoccin commonly is thought to promote secondary solute transport via an increase in electrical driving force which follows the enhancement of primary, “electrogenic” H+ extrusion by the plant plasma membrane H+-ATPase. However, previous electrical studies ofVicia faba L. guard cells in FC (Blatt, 1988, Planta174, 187) demonstrated, in addition to a limited rise in pump current, appreciable declines in membrane conductance near and positive to the free-running membrane potential (Vm). Much of the current at these potentials could have been carried by outward-rectifying K+ channels which were progressively inactivated in FC. We have examined this possibility in electrical studies, using whole-cell currents measured under voltage clamp to quantitate steadystate and kinetic characteristics of the K+ channels both before and during exposure to FC; channels block in tetraethylammonium chloride was exploited to assess changes in background ‘leak’ currents. The cells showed little evidence of primary pump activity, a fact which further simplified analyses. Under these conditions, outward-directed K+ channel current contributed to charge balance maintainingVm, and adding 10 μM FC on average depolarized (positive-going)Vm. Steady-state current-voltage relations revealed changes both in K+ channel and in leak currents underlying the voltage response. Changes in the leak were variable, but on average the leak equilibrium potential was shifted (+)19 mV and leak conductance declined by 21% over 30–40 min in FC. Potassium currents were inactivated irreversibly and with halftimes (current maxima) of 6.2–10.7 min. Inactivation was voltage-dependent, so that the activation (“gating”) potential for the current was shifted, positive-going, with time in FC. Channel gating kinetics, inferred from the macroscopic currents, were also affected; current rise at positive potentials accelerated 4.5-fold and more, but in a manner apparently independent of voltage and extracellular potassium concentration. Current decay at negative potentials was quickened, also. These results identify the outward-rectifying K+ channels as one site of action for FC at a higher plant cell membrane; they complete the link introduced in the preceding paper between K+ channel current, K+(86Rb+) flux and irreversible cation uptake in the toxin. The data also offer some insights toward a kinetic description of channel gating. Finally, they provide a vehicle for interpreting FC-induced changes in K+ and net H+ flux, and in membrane potential without the necessity for postulating gross changes in H+ pumping.

Key words

Current-voltage (I–V) relationship Fusicoccin action H+ pump Membrane potential Potassium channel (voltage gated) Tetraethylammonium chloride Vicia (fusicoccin action) Voltage clamp 

Abbreviations and symbols

EK

equilibrium potential for K+

FC

fusicoccin

Hepes

4-(2-hydroxyethyl)-1-piperazineethan-esulfonic acid

I–V

current-voltage (relationship)

K0+

extracellular K+ (concentration)

TEA

tetraethylammonium chloride

Vm

free-running membrane potential

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References

  1. Assmann, S.M., Simoncini, L., Schroeder, J.I. (1985) Blue light activates electrogenic ion pumping in guard cell protoplasts ofVicia faba. Nature318, 285–287Google Scholar
  2. Bates, G., Goldsmith, M.-H., Godsmith, T. (1983) Membranes of oat cells: the inverse relation between voltage and resistance is not due to H+ pumps. Plant Sci. Lett.30, 279–284Google Scholar
  3. Bertl, A., Felle, H. (1985) Cytoplasmic pH of root hair cells ofSinapis alba recorded by a pH-sensitive microelectrode. Does fusicoccin stimulate the proton pump by cytoplasmic acidification? J. Exp. Bot.36, 1142–1149Google Scholar
  4. Bertl, A., Klieber, H.G., Gradmann, D. (1988) Slow kinetics of a potassium channel inAcetabularia. J. Membr. Biol.102, 141–152Google Scholar
  5. Blatt, M.R. (1987a) Electrical characteristics of stomatal guard cells: the ionic basis of the membrane potential and the consequence of potassium chloride leakage from microelectrodes. Planta170, 272–287Google Scholar
  6. Blatt, M.R. (1987b) Electrical characteristics of stomatal and guard cells: the contribution of ATP-dependent, “electrogenic” transport revealed by current-voltage and differencecurrent-voltage analysis. J. Membr. Biol.98, 257–274Google Scholar
  7. Blatt, M.R. (1988a) Mechanisms of fusicoccin action: a dominant role for secondary transport in a higher-plant cell. Planta174, 187–200Google Scholar
  8. Blatt, M.R. (1988b) Potassium-dependent, bipolar gating of K+ channels in guard cells. J. Membr. Biol.102, 235–246Google Scholar
  9. Blatt, M.R. (1988c) K+ channel gating in guard cells depends on external K+ concentration. Plant Physiol.86, 826AGoogle Scholar
  10. Blatt, M.R., Rodriguez-Navarro, A., Slayman, C.L. (1987) The potassium-proton symport inNeurospora: kinetic control by pH and membrane potential. J. Membr. Biol.98, 169–187Google Scholar
  11. Blatt, M.R., Slayman, C.L. (1987) Role of “active” potassium transport in the regulation of cytoplasmic pH by non-animal cells. Proc. Natl. Acad. Sci.84, 2737–2741Google Scholar
  12. Blum, W., Key, G., Weiler, E.W. (1988) ATPase activity in plasmalemma-rich vesicles isolated by aqueous two-phase partitioning fromVicia faba mesophyll and epidermis: characterization and influence of abscisic acid and fusicoccin. Physiol. Plant.72, 279–287Google Scholar
  13. Briggs, G.E., Hope, A.B., Robertson, R.N. (1961) Electrolytes and Plant Cells. Blackwells, Oxford, UKGoogle Scholar
  14. Brown, P., Outlaw, W.H. Jr. (1982) Effect of fusicoccin on dark14CO2 fixation byVicia faba guard cell protoplasts. Plant Physiol.70, 1700–1703Google Scholar
  15. Castillo, J. del, Katz, B. (1957) Interaction at end-plate receptors between different choline derivatives. Proc. R. Soc. London Ser. B146, 369–381Google Scholar
  16. Cleland, R.E. (1976) Rapid stimulation of K+−H+ exchange by a plant growth hormone. Biochem. Biophys. Res. Commun.69, 333–338Google Scholar
  17. Cleland, R.E., Lomax, T. (1977) Hormonal control of H+-excretion from oat cells. In: Regulation of cell membrane activities in plants, pp. 161–171, Marrè, E., Ciferri, O., eds. Elsevier, AmsterdamGoogle Scholar
  18. Clint, G.M., Blatt, M.R. (1989) Mechanisms of fusicoccin action: Evidence for concerted modulations of secondary K+ transport in a higher-plant cell. Planta178, 495–508Google Scholar
  19. Colquhoun, D., Hawkes, A.G. (1977) Relaxation and fluctuations that flow through drug-operated channels. Proc. R. Soc. London Ser. B199, 231–262Google Scholar
  20. Colombo, R., Bonetti, A., Cerana, R., Lado, P. (1981) Effect of plasmalemma ATPase inhibitors, diethylstillbesterol and orthovanadate, on fusicoccin-induced H+ extrusion in maize roots. Plant Sci. Lett.21, 305–315Google Scholar
  21. Colombo, R., De Michelis, M., Lado, P. (1978) 3-O-methyl glucose uptake stimulation by auxin and by fusicoccin in plant materials and its relationships with proton extrusion. Planta138, 249–256Google Scholar
  22. Felle, H. (1982) Effects of fusicoccin upon membrane potential, resistance and current-voltage characteristics in root hairs ofSinapis alba. Plant Sci. Lett.25, 219–225Google Scholar
  23. Feyerabend, M., Weiler, E.W. (1988) Characterization and localization of fusicoccin-binding sites in leaf tissues ofVicia faba L. probed with a novel radioligand. Planta174, 115–122Google Scholar
  24. Hille, B., Schwarz, W. (1978) Potassium channels as multi-ion, single-file pores. J. Gen. Physiol.72, 409–442Google Scholar
  25. Hodgkin, A., Huxley, A.F., Katz, B. (1952) Measurement of current-voltage relations in the membrane of the giant axon ofLoligo. J. Physiol.116, 424–448Google Scholar
  26. Jennings, I., Rea, P., Leigh, R., Sanders, D. (1988) Quantitative and rapid estimation of H+ fluxes in membrane vesicles. Plant Physiol.86, 125–133Google Scholar
  27. Lass, B., Ullrich-Eberius, C. (1984) Evidence for proton/sulfate cotransport and its kinetics inLemna gibba G1. Planta161, 53–60Google Scholar
  28. Levitan, I.B. (1985) Phosphorylation of ion channels. J. Membr. Biol.87, 177–190Google Scholar
  29. MacRobbie, E.A.C. (1987) Ionic relations of guard cells. In: Stomatal function, pp. 125–162, Zeiger, E., Farquhar, G.D., Cowan, I.R., eds. Stanford University Press, Stanford, Cal. USAGoogle Scholar
  30. Marrè, E. (1979) Fusicoccin: a tool in plant physiology. Annu. Rev. Plant Physiol.30, 273–288Google Scholar
  31. Marrè, E. (1985) Fusicoccin- and hormone-induced changes of H+ extrusion: physiological implications. In: Frontiers of membrane research in agriculture, pp. 439–460, St. John, J., Berlin, E., Jackson, P., eds. Rowman and Allanheld, Ottawa, CanadaGoogle Scholar
  32. Marrè, E., Lado, P., Rasi-Caldogno, F., Colombo, R. (1973) Correlation between cell enlargement in pea internode segments and decrease in the pH of the medium of incubation. II. Effects of inhibitors of respiration, oxidative phosphorylation and protein synthesis. Plant Sci. Lett.1, 185–192Google Scholar
  33. Marrè, E., Lado, P., Rasi-Caldogno, F., Colombo, R., DeMichelis, M.I. (1974) Evidence for the coupling of proton extrusion to K+ uptake in pea internode segments exposed to fusicoccin or auxin. Plant Sci. Lett.3, 365–379Google Scholar
  34. Marquardt, D. (1963) An algorithm for least-squares estimation of nonlinear parameters. J. Soc. Ind. Appl. Math.11, 431–441Google Scholar
  35. Moczydlowski, E., Lucchesi, K., Ravindran, A. (1988) An emerging pharmacology of peptide toxins targeted against potassium channels. J. Membr. Biol.105, 95–111Google Scholar
  36. Rasi-Caldogno, F., DeMichelis, M.I., Pugiarello, M., Marrè, E. (1986) H+-pumping driven by the plasma membrane AT-Pase in membrane vesicles from radish: stimulation by fusicoccin. Plant Physiol.82, 121–125Google Scholar
  37. Reid, R., Field, L., Pitman, M. (1985) Effects of external pH, fusicoccin and butyrate on the cytoplasmic pH in barley root tips measured by31P-NMR spectroscopy. Planta166, 341–347Google Scholar
  38. Roberts, J.K.M., Ray, P.M., Wade-Jardetzky, N., Jardetzky, O. (1981) Extent of intracellular pH changes during H+ extrusion by maize root-tip cells. Planta152, 74–78Google Scholar
  39. Rodriguez-Navarro, A., Blatt, M.R., Slayman, C.L. (1986) A potassium-proton symport inNeurospora crassa. J. Gen. Physiol.87, 649–674Google Scholar
  40. Romani, G., Marrè, M., Bellando, M., Alloatti, G., Marrè, E. (1985) H+ extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments with permeant weak acids. Plant Physiol.79, 734–739Google Scholar
  41. Serrano, E.E., Zeiger, E., Hagiwara, S. (1988) Red light stimulates an electrogenic proton pump inVicia guard cell protoplasts. Proc. Natl. Acad. Sci. USA85, 436–440Google Scholar
  42. Schroeder, J. (1988) Potassium transport properties of the plasma membrane ofVicia faba guard cells. J. Gen. Physiol.92, 667–683Google Scholar
  43. Schroeder, J., Raschke, K., Neher, E. (1987) Voltage dependence of K+ channels in guard cell protoplasts. Proc. Natl. Acad. Sci. USA84, 4109–4112Google Scholar
  44. Shimazaki, K., Iino, M., Zeiger, E. (1986) Blue light-dependent proton extrusion by guard-cell protoplasts ofVicia faba. Nature319, 324–326Google Scholar
  45. Shuster, M.J., Camardo, J.S., Siegelbaum, S.A., Kandel, E.R. (1985) Cyclic AMP-dependent protein kinase closes the serotonin-sensitive K+ channels ofAplysia sensory neurones in cell-free membrane patches. Nature313, 391–395Google Scholar
  46. Stanfield, P.R. (1983) Tetraethylammonium ions and the potassium permeability of excitable cells. Rev. Physiol. Biochem. Pharmacol.97, 1–67Google Scholar
  47. Stelzer, A., Kay, A.R., Wong, R.K.S. (1988) GABAA-receptor function in hippocampal cells is maintained by phosphorylation factors. Science241, 339–341Google Scholar
  48. Stout, R., Cleland, R.E. (1980) Partial characterization of fusicoccin binding to receptor sites on oat root membranes. Plant Physiol.66, 353–359Google Scholar
  49. Stout, R., Johnson, K.D., Rayle, D. (1978) Rapid auxin- and fusicoccin-enhanced Rb+ uptake and malate synthesis inAvena coleoptile sections. Planta139, 35Google Scholar
  50. Stryer, L. (1986) Cyclic GMP cascade of vision. Annu. Rev. Neurosci.9, 87–119Google Scholar
  51. Tester, M. (1988) Pharmacology of K+ channels in the plasmalemma of the green algaChara corallina. J. Membr. Biol.103, 159–169Google Scholar
  52. Ullrich-Eberius, C., Novacky, A., van Bel, A.J. (1984) Phosphate uptake inLemna gibba G1: energetics and kinetics. Planta161, 46–52Google Scholar
  53. van Bel, A.J., Ammerlaan, A. (1981) Light-promoted diffusional amino acid efflux fromCommelina leaf disks: indirect control by proton pump activities. Planta152, 115–123Google Scholar
  54. Vandenberg, C.A. (1987) Inward rectification of potassium channel in cardiac ventricular cells depends on internal magnesium ions. Proc. Natl. Acad. Sci. USA84, 2560–2564Google Scholar
  55. Yatani, A., Codina, J., Brown, A., Birnbaumer, L. (1987) Direct activation of mammalian atrial muscarinic potassium channels by GTP regulatory proteinG K. Science235, 207–211Google Scholar
  56. Zucker, R.S. (1981) Tetraethylammonium contains an impurity which alkalizes cytoplasm and reduces calcium buffering in neurones. Brain Res.208, 473–478Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Michael R. Blatt
    • 1
  • Gill M. Clint
    • 1
  1. 1.Botany SchoolUniversity of CambridgeCambridgeUK

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