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

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).