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In planta AKT2 subunits constitute a pH- and Ca2+-sensitive inward rectifying K+ channel

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Abstract

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.

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Abbreviations

mGFP:

Modified green-fluorescent protein

HEK:

Human embryonic kidney epithelial

Agroinfiltration:

Infiltration of agrobacteria

V5:

Epitope tag derived from the P and V proteins of the paramyxovirus of simian virus 5 (SV5)

CHO:

Chinese hamster ovarian

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Acknowledgments

We thank Tina Romeis (Biochemistry of Plants, Department of Biology, Free University of Berlin, Germany), Sean Chapman and Karl Oparka (Scottish Crop Research Institute, Dundee, UK) for providing N. benthamiana seeds and Agrobacterium tumefaciens GV3101 strain carrying 19K gene, and introducing us into the agroinfiltration technique. For critical reading of the manuscript we are grateful to Irene Marten. This work was funded by DFG SFB 478 grants.

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Authors and Affiliations

  1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs Institute of Biosciences, University of Wuerzburg, Julius-von-Sachs-Platz 2, 97082, Wuerzburg, Germany

    Andreas Latz, Susanne Fischer, Peter Ache, Dirk Becker, Rosalia Deeken & Rainer Hedrich

  2. School of Biological Sciences, University of Wales Bangor, Bangor, Deiniol Road, Gwynedd, LL57 2UW, Wales, UK

    Natalya Ivashikina

  3. Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, 277-8562, Japan

    Toshio Sano

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  1. Andreas Latz
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  2. Natalya Ivashikina
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  3. Susanne Fischer
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  4. Peter Ache
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  5. Toshio Sano
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  6. Dirk Becker
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  7. Rosalia Deeken
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  8. Rainer Hedrich
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Correspondence to Rainer Hedrich.

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Andreas Latz and Natalya Ivashikina contributed equally to this work.

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Latz, A., Ivashikina, N., Fischer, S. et al. In planta AKT2 subunits constitute a pH- and Ca2+-sensitive inward rectifying K+ channel. Planta 225, 1179–1191 (2007). https://doi.org/10.1007/s00425-006-0428-4

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  • DOI: https://doi.org/10.1007/s00425-006-0428-4

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