Skip to main content
SpringerLink
Log in

Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe

Effects of other anions, membrane potential, and transport inhibitors

  • Published:
Planta Aims and scope Submit manuscript

Abstract

The characteristics of Cl− transport in isolated tonoplast vesicles from red-beet (Beta vulgaris L.) storage tissue have been investigated using the Cl−-sensitive fluorescent probe, 6-methoxy-1-(3-sulfonatopropyl)-quinolinium (SPQ). The imposition of (inside) positive diffusion potentials, generated with K+ and valinomycin, increased the initial rate of Cl− transport, demonstrating that Cl− could be electrically driven into the vesicles. Chloride influx was unaffected by SO 2-4 , but was competitively blocked by NO −3 , indicating that both Cl− and NO −3 may be transported by the same porter. In some preparations, increases in free-Ca2+ concentration from 10−8 to 10−5 mol·dm−3 caused a significant decrease in Cl− influx, which may indicate that cytosolic Ca2+ concentration has a role in controlling Cl− fluxes at the tonoplast. However, this effect was only seen in about 50% of membrane preparations and some doubt remains over its physiological significance. A range of compounds known to block anion transport in other systems was tested, and some partially blocked Cl− transport. However, many of these inhibitors interfered with SPQ fluorescence and so only irreversible effects could be tested. The results are discussed in the context of recent advances made using the patch-clamp technique on isolated vacuoles.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

BTP:

1,3-bis[tris(hydroxymethyl)-methylamino]propane

DTT:

dithiothreitol

EDTA:

ethylenediaminetetraacetic acid

Δψ :

membrane potential

ΔpH:

pH gradient

SPQ:

6-methoxy-1-(3-sulfonatopropyl)quinolinium

Tricine:

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl] glycine

References

  • Bates, G.W., Goldsmith, M.H.M., Goldsmith, T.H. (1982) Separation of tonoplast and plasma membrane potential and resistance in cells of oat coleoptiles. J. Membr. Biol. 66, 15–23

    Google Scholar 

  • Bennett, A.B., Spanswick, R.M. (1983) Optical measurement of ΔpH and Δψ in corn root membrane vesicles: kinetic analysis of Cl− effects on a proton-translocating ATPase. J. Membr. Biol. 71, 95–107

    Google Scholar 

  • Blumwald, E., Poole, R.J. (1985) Na+/H+ antiport in isolated tonoplast vesicles from storage tissue of Beta vulgaris L. Plant Physiol. 78, 163–167

    Google Scholar 

  • Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilising the principle of dye-binding. Anal. Biochem. 72, 248–256

    Article  CAS  PubMed  Google Scholar 

  • Bretag, A.H. (1987) Muscle chloride channels. Physiol. Rev. 67, 621–724

    Google Scholar 

  • Bretag, A.H., Dawe, S.R., Kerr, D.I.B., Moskwa, A.G. (1980) Myotonia as a side effect of diuretic action. Br. J. Pharmacol. 71, 467–471

    Google Scholar 

  • Brule, G., Hardecour, G., Idaiha, H., Guilbault, P. (1983) Inhibition par une substance amphiphile, l'acide niflumique, de la rectification dans le sens entrant de la fibre musculaire de crustacea. Arch. Int. Physiol. Biochem. 91, 269–277

    Google Scholar 

  • Cabantchik, Z.I., Knauf, P.A., Rothstein, A. (1978) The anion transport system of the red blood cell. The role of membrane protein evaluated by the use of ‘probes’. Biochim. Biophys. Acta 515, 239–302

    Google Scholar 

  • Chen, P.Y., Verkman, A.S. (1988) Sodium-dependent chloride transport in basolateral membrane vesicles isolated from rabbit proximal tubule. Biochemistry 27, 655–660

    Google Scholar 

  • Chen, P.Y., Illsley, N.P., Verkman, A.S. (1988) Renal brush border chloride transport mechanisms characterised using a fluorescent indicator. Am. J. Physiol. 254, F114-F120

    Google Scholar 

  • Churchill, K.A., Sze, H. (1984) Anion-sensitive, H+-pumping ATPase of oat roots. Direct effects of Cl−, NO -3 , and a disulfonic stilbene. Plant Physiol. 87, 490–497

    Google Scholar 

  • Coyaud, L., Kurkdjian, A., Kado, R., Hedrich, R. (1987) Ion channels and ATP-driven pumps involved in ion transport across the tonoplast of sugarbeet vacuoles. Biochim. Biophys. Acta 902, 263–268

    Google Scholar 

  • Cupoletti, J., Sachs, G. (1986) Regulation of gastric acid secretion via modulation of a chloride conductance. J. Biol. Chem. 23, 14952–14959

    Google Scholar 

  • Dechecci, M.C., Cabrini, G. (1988) Chloride conductance in membrane vesicles from human placenta using a fluorescent probe. Implications for cystic fibrosis. Biochim. Biophys. Acta 945, 113–120

    Google Scholar 

  • Falke, J.J., Chan, S.I. (1986a) Molecular mechanisms of band 3 inhibitors. 1. Transport site inhibitors. Biochemistry 25, 7888–7894

    Google Scholar 

  • Falke, J.J., Chan, S.I.. (1986b) Molecular mechanisms of band 3 inhibitors. 2. Channel blockers. Biochemistry 25, 7895–7898

    Google Scholar 

  • Falke, J.J., Chan, S.I. (1986c) Molecular mechanisms of band 3 inhibitors. 3. Translocation inhibitors. Biochemistry 25, 7899–7906

    Google Scholar 

  • Flowers, T.J.. (1988) Chloride as a nutrient and as an osmoticum. In: Advances in plant nutrition, vol. 3, pp. 55–78, Tinker, P.B., Läuchli, A., eds. Praeger, New York

    Google Scholar 

  • Garlid, K.D., Beavis, A.D. (1986) Evidence for the existence of an inner membrane anion channel in mitochondria. Biochim. Biophys. Acta 853, 187–204

    Google Scholar 

  • Hedrich, R., Kurkdjian, A. (1988) Characterisation of an anionpermeable channel from sugar beet vacuoles: effect of inhibitors. EMBO J. 7, 3661–3666

    Google Scholar 

  • Hedrich, R., Neher, E. (1987) Cytoplasmic calcium regulates voltagedependent ion channels in plant vacuoles. Nature 329, 833–836

    Google Scholar 

  • Hedrich, R., Barbier-Brygoo, H., Felle, H., Flügge, U.I., Lüttge, U., Maathuis, F.J.M., Marx, S., Prins, H.B.A., Raschke, K., Schnabl, H., Schroeder, J.I., Struve, I., Taiz, L., Zeigler, P. (1988) General mechanisms for solute transport across the tonoplast of plant vacuoles: a patch-clamp survey of ion channels and proton pumps. Bot. Acta 101, 7–13

    Google Scholar 

  • Illsley, N.P., Verkman, A.S. (1987) Membrane chloride transport measured using a chloride-sensitive fluorescent probe. Biochemistry 26, 1215–1219

    Google Scholar 

  • Jennings, I.R., Rea, P.A., Leigh, R.A., Sanders, D. (1988) Quantitative and rapid estimation of H+ fluxes in membrane vesicles: Software for analysis of fluorescence quenching and relaxation. Plant Physiol. 86, 1257–1263

    Google Scholar 

  • Kaestner, K.H., Sze, H. (1987) Potential-dependent anion transport in tonoplast vesicles from oat roots. Plant Physiol. 83, 483–489

    Google Scholar 

  • Lunevsky, V.Z., Zherelova, O.M., Vostrikov, I.Y., Berestovsky, G.N. (1983) Excitation of characean cell membranes as a result of activation of calcium and chloride channels. J. Membr. Biol. 72, 43–58

    Google Scholar 

  • MacRobbie, E.A.C. (1970) The active transport of ions in plant cells. Quart. Rev. Biophys. 3, 251–294

    Google Scholar 

  • Nelson, D.J., Tang, J.M., Palmer, L.G. (1984) Single-channel recordings of apical membrane chloride conductance in A6 epithelial cells. J. Membr. Biol. 80, 81–89

    Google Scholar 

  • Nichols, D.G. (1982) Bioenergetics: an introduction to the chemiosmotic theory. Academic Press, London

    Google Scholar 

  • Pope, A.J., Leigh, R.A. (1987) Some characteristics of anion transport at the tonoplast of oat roots, determined from the effects of anions on pyrophosphate-dependent proton transport. Planta 172, 91–100

    Google Scholar 

  • Pope, A.J., Leigh, R.A. (1988) The use of a chloride-sensitive fluorescent probe to measure chloride transport in isolated tonoplast vesicles. Planta 176, 451–460

    Google Scholar 

  • Pope, A.J., Jennings, I.R., Sanders, D., Leigh, R.A. (1990) Characterisation of Cl− transport in vacuolar membrane vesicles using a chloride-sensitive fluorescent probe. Reaction kinetic models for voltage- and concentration-dependence of Cl− flux. J. Membr. Biol. (in press)

  • Rea, P.A., Poole, R.J. (1985) Proton-translocating inorganic pyrophosphatase in red beet (Beta vulgaris L.) tonoplast vesicles. Plant Physiol. 77, 46–52

    Google Scholar 

  • Rea, P.A., Leigh, R.A., Sanders, D. (1987) Wash activated Na +/ H+ exchange in tonoplast vesicles from Beta vulgaris storage root disks. In: Plant vacuoles: their importance in solute compartmentation and their applications in plant biotechnology, pp. 227–233, Marin, B., ed. Plenum Press, New York

    Google Scholar 

  • Rudel, R., Senges, J. (1972) Experimental myotonia in mammalian skeletal muscle: change in membrane properties. Pflugers Arch. 331, 324–334

    Google Scholar 

  • Schroeder, J.I., Hedrich, R. (1989) Involvement of ion channels and active transport in osmoregulation and signalling of higher plant cells. Trends Biochem. Sci. 14, 187–192

    Google Scholar 

  • Schumaker, K.H., Sze, H. (1987) Decrease in pH gradients in tonoplast vesicles by Cl− and NO -3 : evidence for H+-coupled anion transport. Plant Physiol. 83, 490–496

    Google Scholar 

  • Sze, H. (1985) H+-translocating ATPases: advances using membrane vesicles. Annu. Rev. Plant Physiol. 36, 175–208

    Google Scholar 

  • Ticku, M.K., Olsen, R.W. (1977) γ-aminobutyric acid-stimulated chloride permeability in crayfish muscle. Biochim. Biophys. Acta 464, 519–529

    Google Scholar 

  • Tyerman, S.D., Findlay, G.P. (1989) Current-voltage curves of single Cl− channels which coexist with two types of K+ channel in the tonoplast of Chara corallina. J. Exp. Bot. 40, 105–117

    Google Scholar 

  • Tyerman, S.D., Findlay, G.P., Paterson, G.J. (1986) Inward membrane current in Chara inflata. II. Effects of pH, Cl− channel blockers and NH +4 , and significance of the hyperpolarised state. J. Membr. Biol. 89, 153–161

    Google Scholar 

  • Welsh, M.J. (1983) Inhibition of chloride secretion by furosemide on canine tracheal epithelium. J. Membr. Biol. 71, 219–226

    Google Scholar 

  • Welsh, M.J. (1984) Anthracene-9-carboxylic acid inhibits an apical membrane chloride conductance in canine tracheal epithelium. J. Membr. Biol. 78, 61–71

    Google Scholar 

  • Wolfbeis, O.S., Urbano, E. (1982) Synthesis of fluorescent dyes. XIV. Standards for fluorescence measurements in the near-neutral pH range. J. Heterocyclic Chem. 19, 841–843

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Biochemistry and Physiology Department, AFRC Institute of Arable Crops Resarch, Rothamsted Experimental Station, AL5 2JQ, Harpenden, Hertfordshire, UK

    Andrew J. Pope & Roger A. Leigh

Authors
  1. Andrew J. Pope
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Roger A. Leigh
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pope, A.J., Leigh, R.A. Characterisation of chloride transport at the tonoplast of higher plants using a chloride-sensitive fluorescent probe. Planta 181, 406–413 (1990). https://doi.org/10.1007/BF00195895

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00195895

Key words

Search

Navigation