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TPK1, a Ca2+-regulated Arabidopsis vacuole two-pore K+ channel is activated by 14-3-3 proteins

Authors

  • A. Latz,

    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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  • D. Becker,

    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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  • M. Hekman,

    1. Department of Physiological Chemistry, Biocenter, University of Würzburg, Germany
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  • T. Müller,

    1. Botanical Institute I, University of Karlsruhe (TH), Kaiserstrasse 12, 76128 Karlsruhe, Germany
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  • D. Beyhl,

    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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  • I. Marten,

    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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  • C. Eing,

    1. Institute for Medical Radiation and Cell Research, University of Würzburg, Würzburg 97078, Germany
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  • A. Fischer,

    1. Department of Physiological Chemistry, Biocenter, University of Würzburg, Germany
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  • M. Dunkel,

    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
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  • A. Bertl,

    1. Institute for Medical Radiation and Cell Research, University of Würzburg, Würzburg 97078, Germany
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  • U. R. Rapp,

    1. Department of Physiological Chemistry, Biocenter, University of Würzburg, Germany
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  • R. Hedrich

    Corresponding author
    1. Department of Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, Biocenter, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
      (fax +49 931/888 6157; e-mail hedrich@botanik.uni-wuerzburg.de).
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(fax +49 931/888 6157; e-mail hedrich@botanik.uni-wuerzburg.de).

Summary

The vacuole represents a pivotal plant organelle for management of ion homeostasis, storage of proteins and solutes, as well as deposition of cytotoxic compounds. Ion channels, pumps and carriers in the vacuolar membrane under control of cytosolic factors provide for ionic and metabolic homeostasis between this storage organelle and the cytoplasm. Here we show that AtTPK1 (KCO1), a vacuolar membrane localized K+ channel of the TPK family, interacts with 14-3-3 proteins (general regulating factors, GRFs). Following in planta expression TPK1 and GRF6 co-localize at the vacuolar membrane. Co-localization of wild-type TPK1, but not the TPK1-S42A mutant, indicates that phosphorylation of the 14-3-3 binding motif of TPK1 represents a prerequisite for interaction. Pull-down assays and surface plasmon resonance measurements revealed GRF6 high-affinity interaction with TPK1. Following expression of TPK1 in yeast and isolation of vacuoles, patch-clamp studies identified TPK1 as a voltage-independent and Ca2+-activated K+ channel. Addition of 14-3-3 proteins strongly increased the TPK1 activity in a dose-dependent manner. However, an inverse effect of GRF6 on the activity of the slow-activating vacuolar (SV) channel was observed in mesophyll vacuoles from Arabidopsis thaliana. Thus, TPK1 seems to provide for a Ca2+- and 14-3-3-sensitive mechanism capable of controlling cytoplasmic potassium homeostasis in plants.

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