A procedure for localisation and electrophysiological characterisation of ion channels heterologously expressed in a plant context.
Plant Methods. 2005-01-01; 1(1): 14
DOI: 10.1186/1746-4811-1-14
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1. Plant Methods. 2005 Dec 19;1:14. doi: 10.1186/1746-4811-1-14.
A procedure for localisation and electrophysiological characterisation of ion
channels heterologously expressed in a plant context.
Hosy E(#)(1)(2), Duby G(#)(1)(3), Véry AA(1), Costa A(1)(4), Sentenac H(1),
Thibaud JB(1).
Author information:
(1)Biochimie et Physiologie Moléculaires des Plantes, UMR 5004,
Agro-M/CNRS/INRA/UM2, F-34060 Montpellier Cedex 1, France.
(2)Present address: Laboratoire de Biophysique Moléculaire et Cellulaire, UMR
5090, CEA-DRDC-BMC, 17 rue des Martyrs, F-38054 Grenoble Cedex 9, France.
(3)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.
(4)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.
(#)Contributed equally
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.
CONCLUSION: 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.
DOI: 10.1186/1746-4811-1-14
PMCID: PMC1352354
PMID: 16359560