In planta AKT2 subunits constitute a pH- and Ca2+-sensitive inward rectifying K+ channel.
Heterologous expression of plant genes in yeast and animal cells represents a common approach to study plant ion channels. When expressed in Xenopus oocytes and COS cells the Arabidopsis Shaker-like K+ channel, AKT2 forms a weakly voltage-dependent channel, blocked by Ca2+ and protons. Channels with these characteristics, however, were not found in AKT2-expressing Arabidopsis cell types. To understand this phenomenon, we employed Agrobacterium-mediated transient transformation to functionally characterise Arabidopsis thaliana channels in Nicotiana benthamiana mesophyll cells. In this expression system we used AtTPK4 as a control for voltage-independent A. thaliana channels. Agrobacteria harbouring GFP-tagged constructs with the coding sequences of AKT2 and AtTPK4 were infiltrated into intact tobacco leaves. With quantitative RT-PCR analyses channel transcripts of AKT2 and AtTPK4 were determined in transformed leaves. These results were confirmed by Western blots with V5 epitope-tagged AKT2 and AtTPK4 proteins, showing that the channel protein was indeed synthesised. For functional analysis of these channels, mesophyll protoplasts were isolated from infiltrated leaf sections. Patch-clamp studies revealed that AKT2 channels in mesophyll protoplasts retained Ca2+ and pH sensitivity, characteristics of the heterologously expressed protein, but displayed pronounced differences in voltage-dependence and kinetics. AKT2-transformed mesophyll cells, displayed inward-rectifying, rather than voltage-independent K+ channels, initially characterised in AKT2-expressing animal cells. In contrast, AtTPK4 showed the same electrophysiological characteristics both, in oocytes and plant cells. Our data suggest that heterologous systems do not always possess all regulatory components for functional expression of plant channels. Therefore, transient expression of plant proteins in planta provides an additional research tool for rapid biophysical analysis of plant ion channels.
Latz
Andreas
A
Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082 Wuerzburg, Germany.
Ivashikina
Natalya
N
Fischer
Susanne
S
Ache
Peter
P
Sano
Toshio
T
Becker
Dirk
D
Deeken
Rosalia
R
Hedrich
Rainer
R
D017360:Arabidopsis / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000502:physiology*; D018744:DNA, Plant / Q000235:genetics; D018408:Patch-Clamp Techniques; D018515:Plant Leaves / Q000166:cytology / Q000502:physiology; D030821:Plants, Genetically Modified; D015221:Potassium Channels / Q000235:genetics / Q000502:physiology*; D011523:Protoplasts / Q000502:physiology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012231:Rhizobium / Q000235:genetics; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000502:physiology*; D014158:Transcription, Genetic
C097485:AKT2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D018744:DNA, Plant; D015221:Potassium Channels