A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.
BACKGROUND
In silico analyses based on sequence similarities with animal channels have identified a large number of plant genes likely to encode ion channels. The attempts made to characterise such putative plant channels at the functional level have most often relied on electrophysiological analyses in classical expression systems, such as Xenopus oocytes or mammalian cells. In a number of cases, these expression systems have failed so far to provide functional data and one can speculate that using a plant expression system instead of an animal one might provide a more efficient way towards functional characterisation of plant channels, and a more realistic context to investigate regulation of plant channels.


RESULTS
With the aim of developing a plant expression system readily amenable to electrophysiological analyses, we optimised experimental conditions for preparation and transformation of tobacco mesophyll protoplasts and engineered expression plasmids, that were designed to allow subcellular localisation and functional characterisation of ion channels eventually in presence of their putative (possibly over-expressed) regulatory partners. Two inward K+ channels from the Shaker family were functionally expressed in this system: not only the compliant KAT1 but also the recalcitrant AKT1 channel, which remains electrically silent when expressed in Xenopus oocytes or in mammalian cells.


CONCLUSIONS
The level of endogenous currents in control protoplasts seems compatible with the use of the described experimental procedures for the characterisation of plant ion channels, by studying for instance their subcellular localisation, functional properties, structure-function relationships, interacting partners and regulation, very likely in a more realistic context than the classically used animal systems.
Hosy
E
E
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.|Present address: Laboratoire de Biophysique Moléculaire et Cellulaire, UMR 5090, CEA-DRDC-BMC, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
Duby
G
G
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.|Present address: Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université Catholique Louvain, Place Croix du Sud, 5-15, 1348 Louvain-la-Neuve, Belgium.
Véry
A-A
AA
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.
Costa
A
A
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.|Present address: Division of Biology, Cell and Developmental Biology Section, and Center for Molecular Genetics, University of California San Diego, CA 92093-0116 La Jolla, USA.
Sentenac
H
H
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.
Thibaud
J-B
JB
Biochimie et Physiologie Moléculaires des Plantes, UMR 5004, Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.