The Study of (Plant) Ion Channels Reconstituted in Planar Lipid Bilayers

  • Henk Miedema
Part of the Springer Lab Manual book series (SLM)

Abstract

The development of the patch-clamp technique by Neher and Sakmann (1976) opened the door to the study of single ion channels. Initially performed on animal cells, 8 years later the technique was successfully applied to plant cells as well (Moran et al. 1984). One reason for the delay in applying patch-clamp to plant cells is the presence of a cell wall which has to be removed before the plant protoplast can be patch-clamped (Takeda et al. 1985). Today, patch-clamp is an almost standard technique in laboratories around the world specializing in plant cell electrophysiology. Despite the loss of the cell wall, during a patch-clamp experiment the ion channel is studied in its more or less native membrane environment. An alternative strategy to study ion channels is a method based on the reconstitution of the isolated, purified channel in an artificial membrane system, such as a liposome or a planar lipid bilayer. Obviously, these techniques are especially useful in cases in which the particular membrane is inaccessible to the patch-clamp pipette (Miller 1983). The small size of liposomes and the high turnover rate of ion channels, however, limit the use of liposomes for studies of channel mediated transport (Miller 1983). The performance of electrophysiological measurements requires either enlargement of the liposomes to a size suitable for patch-clamping (Tank and Miller 1983) or the reconstitution of the channel into a so-called planar lipid bilayer (PLB). The PLB technique allows the study of ion channels on the single channel level under precise, well defined experimental conditions and has been appplied succesfully to study ion channels originating from animal cell membranes and endomembranes (see references in Labarca and Latorre 1992; Coronado et al. 1992). White and Tester (1992) were the first to reconstitute plant plasma membrane channels in a PLB. At the same time, Klughammer et al. (1992a,b) applied the PLB technique to study ion channels in the plant vacuolar membrane. Here we describe a methodology to set up such a PLB system and show measurements obtained after incorporation of plant vacuolar channels in a PLB. The more interested reader is referred to the superb textbooks by Hanke and Schlue (1993) and Miller (1986) and to Labarca and Latorre (1992).

Keywords

Cholesterol Permeability Agar Cage Hexane 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Coronado R, Kawano S, Lee CJ, Valdivia C, Valdivia HH (1992) Planar Bilayer recording of ryanodine receptors of sarcoplasmic reticulum. In: Rudy B, Iverson LE (eds) Methods in enzymology, Academic, San Diego, 207: 699–707Google Scholar
  2. Ehrlich BE (1992) Incorporation of ion channels in planar lipid bilayers. In: Fozzard et al. (eds) The heart and cardiovascular system. Raven, New York, pp 551–560Google Scholar
  3. Hanke W, Schlue WR (1993) Planar lipid bilayers. Academic, LondonGoogle Scholar
  4. Klughammer B, Benz R, Betz M, Thume M, Dietz K-J (1992a) Reconstitution of vacuolar ion channels into planar lipid bilayers. Biochem Biophys Acta 1104: 308–316PubMedCrossRefGoogle Scholar
  5. Klughammer B, Betz M, Benz R, Dietz K-J (1992b) Partial purification of a potassium channel with low permeability for sodium from tonoplast membranes of Hordeum vulgare cv. Gerbel. J Membrane Biol 128: 17–25Google Scholar
  6. Labarca P, Latorre R (1992) Insertion of ion channels into planar lipid bilayers by vesicle fusion. In: Rudy B, Iverson LE (eds) Methods in enzymology, Academic, San Diego, 207: 447–463Google Scholar
  7. Miller C (1983) Reconstitution of ion channels in planar lipid bilayer membranes: a five-year progress report. Comments Mol Cell Biophys 1: 413–428Google Scholar
  8. Miller C (1986) Ion channel reconstitution. Plenum, New YorkGoogle Scholar
  9. Moran N, Ehrenstein G, Iwasa K, Bare C, Mischke D (1984) Ion channels in plasma. lemma of wheat protoplasts. Science 226: 835–838PubMedCrossRefGoogle Scholar
  10. Neher E, Sakmann B (1976) Singel channel currents recorded from membrane of denervated frog muscle fibres. Nature 260: 779–802CrossRefGoogle Scholar
  11. Pineros M, Tester M (1995) Characterization of a voltage dependent Cat+ selective. channel from wheat roots. Planta 195: 478–488CrossRefGoogle Scholar
  12. Rea PA, Poole RJ (1985) Proton translocating inorganic pyrophosphatase in red beet (Beta vulgaris L.) tonoplast vesicles. Plant Physiol 77: 46–52PubMedCrossRefGoogle Scholar
  13. Schulz-Lessdorf B, Hedrich R (1995) Protons and calcium modulate SV-type channels in the vacuolar-lysosomal compartment; channel interaction with calmodulin inhibitors. Planta 197: 655–671CrossRefGoogle Scholar
  14. Takeda K, Kurkdjian AC, Kado RT (1985) Ionic channels, ion transport and plant cell membranes: potential applications of the patch-clamp technique. Protoplasma 127: 147–162CrossRefGoogle Scholar
  15. Tank DW, Miller C (1983) Patch-clamped liposomes: Recording reconstituted ion channels. In: Sakmann B, Neher E (eds) Single channel recording. Plenum, New York, pp 91–106CrossRefGoogle Scholar
  16. White PJ, Tester, MA (1992) Potassium channels from the plasma membrane of rye. roots characterized following incorporation into planar lipid bilayers. Planta 186: 188–202CrossRefGoogle Scholar
  17. White PJ, Tester MA (1994) Using planar lipid bilayers to study plant ion channels. Physiologia Plantarum 91: 770–774CrossRefGoogle Scholar
  18. White PJ, Smahel M, Thiel G (1993) Characterization of ion channels from Acetabularia plasma membrane in planar lipid bilayers. J Membrane Biol 133: 145–160CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • Henk Miedema

There are no affiliations available

Personalised recommendations