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The Arabidopsis AtPP2CA Protein Phosphatase Inhibits the GORK K+ Efflux Channel and Exerts a Dominant Suppressive Effect on Phosphomimetic-activating Mutations*

  • Author Footnotes
    1 Both authors contributed equally to this work.
    ,
    Author Footnotes
    2 Present address: Institute of Molecular, Cellular, and Systems Biology, College of Medical, Veterinary, and Life Sciences, Bower Bldg., University of Glasgow, Glasgow G12 8QQ, UK.
    Cécile Lefoulon
    Footnotes
    1 Both authors contributed equally to this work.
    2 Present address: Institute of Molecular, Cellular, and Systems Biology, College of Medical, Veterinary, and Life Sciences, Bower Bldg., University of Glasgow, Glasgow G12 8QQ, UK.
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Martin Boeglin
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Bertrand Moreau
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Anne-Aliénor Véry
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Wojciech Szponarski
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Myriam Dauzat
    Affiliations
    the Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux, INRA/SupAgro, UMR 759, 2 Place Viala, 34060 Montpellier Cedex, France
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  • Erwan Michard
    Footnotes
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Isabelle Gaillard
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Isabelle Chérel
    Correspondence
    To whom correspondence should be addressed: Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/Université de Montpellier, 2 Place Viala, 34060 Montpellier Cedex. E-mail: .
    Affiliations
    From the Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, CNRS/INRA/SupAgro/UM2, Unité Mixte de Recherche (UMR) 5004, 2 Place Viala, 34060 Montpellier Cedex, France and
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  • Author Footnotes
    * This work was supported by Agence Nationale pour la Recherche contracts PUMPKin ANR-08-BLAN-01 312133 and Sweetkaligrape ANR-14-CE20-0002. The authors declare that they have no conflicts of interest with the contents of this article.
    1 Both authors contributed equally to this work.
    2 Present address: Institute of Molecular, Cellular, and Systems Biology, College of Medical, Veterinary, and Life Sciences, Bower Bldg., University of Glasgow, Glasgow G12 8QQ, UK.
    3 Present address: Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6 P-2780-156 Oeiras, Portugal.
Open AccessPublished:January 22, 2016DOI:https://doi.org/10.1074/jbc.M115.711309
      The regulation of the GORK (Guard Cell Outward Rectifying) Shaker channel mediating a massive K+ efflux in Arabidopsis guard cells by the phosphatase AtPP2CA was investigated. Unlike the gork mutant, the atpp2ca mutants displayed a phenotype of reduced transpiration. We found that AtPP2CA interacts physically with GORK and inhibits GORK activity in Xenopus oocytes. Several amino acid substitutions in the AtPP2CA active site, including the dominant interfering G145D mutation, disrupted the GORK-AtPP2CA interaction, meaning that the native conformation of the AtPP2CA active site is required for the GORK-AtPP2CA interaction. Furthermore, two serines in the GORK ankyrin domain that mimic phosphorylation (Ser to Glu) or dephosphorylation (Ser to Ala) were mutated. Mutations mimicking phosphorylation led to a significant increase in GORK activity, whereas mutations mimicking dephosphorylation had no effect on GORK. In Xenopus oocytes, the interaction of AtPP2CA with “phosphorylated” or “dephosphorylated” GORK systematically led to inhibition of the channel to the same baseline level. Single-channel recordings indicated that the GORK S722E mutation increases the open probability of the channel in the absence, but not in the presence, of AtPP2CA. The dephosphorylation-independent inactivation mechanism of GORK by AtPP2CA is discussed in relation with well known conformational changes in animal Shaker-like channels that lead to channel opening and closing. In plants, PP2C activity would control the stomatal aperture by regulating both GORK and SLAC1, the two main channels required for stomatal closure.

      Introduction

      The plant clade A protein phosphatases 2C (PP2Cs)
      The abbreviations used are: PP2C, protein phosphatase 2C; ABA, abscisic acid; PYR, pyrabactin resistance; PYL, PYR1-like; RCAR, regulatory component of the abscisic acid receptor; CPK, calcium-dependent protein kinase; CNBD, cyclic nucleotide-binding domain; CIPK, calcineurin B-like interacting protein kinase; GORK, guard cell outward rectifying channel; KAT, K+ channel in Arabidopsis thaliana.
      are Mg2+- and Mn2+-dependent serine/threonine phosphatases that were first identified as components of the abscisic acid (ABA) signal transduction pathway (
      • Leung J.
      • Bouvier-Durand M.
      • Morris P.C.
      • Guerrier D.
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      Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.
      ,
      • Meyer K.
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      • Grill E.
      A protein phosphatase 2C involved in ABA signal transduction in Arabidopsis thaliana.
      ). The clade A APP2C members in Arabidopsis are mostly known as negative regulators of ABA signaling (
      • Schweighofer A.
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      • Meskiene I.
      Plant PP2C phosphatases: emerging functions in stress signaling.
      ). Among this clade, ABI1, ABI2 (
      • Merlot S.
      • Gosti F.
      • Guerrier D.
      • Vavasseur A.
      • Giraudat J.
      The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway.
      ), HAB1 (
      • Leonhardt N.
      • Kwak J.M.
      • Robert N.
      • Waner D.
      • Leonhardt G.
      • Schroeder J.I.
      Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.
      ), and AtPP2CA (
      • Sheen J.
      Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants.
      ,
      • Kuhn J.M.
      • Boisson-Dernier A.
      • Dizon M.B.
      • Maktabi M.H.
      • Schroeder J.I.
      The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA.
      • Yoshida T.
      • Nishimura N.
      • Kitahata N.
      • Kuromori T.
      • Ito T.
      • Asami T.
      • Shinozaki K.
      • Hirayama T.
      ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs.
      ) are well characterized. These proteins are involved in ABA-regulated germination (
      • Kuhn J.M.
      • Boisson-Dernier A.
      • Dizon M.B.
      • Maktabi M.H.
      • Schroeder J.I.
      The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA.
      • Yoshida T.
      • Nishimura N.
      • Kitahata N.
      • Kuromori T.
      • Ito T.
      • Asami T.
      • Shinozaki K.
      • Hirayama T.
      ABA-hypersensitive germination3 encodes a protein phosphatase 2C (AtPP2CA) that strongly regulates abscisic acid signaling during germination among Arabidopsis protein phosphatase 2Cs.
      ,
      • Leung J.
      • Merlot S.
      • Giraudat J.
      The Arabidopsis ABSCISIC ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous protein phosphatases 2C involved in abscisic acid signal transduction.
      • Saez A.
      • Apostolova N.
      • Gonzalez-Guzman M.
      • Gonzalez-Garcia M.P.
      • Nicolas C.
      • Lorenzo O.
      • Rodriguez P.L.
      Gain-of-function and loss-of-function phenotypes of the protein phosphatase 2C HAB1 reveal its role as a negative regulator of abscisic acid signalling.
      ). Of these, the ABI1, ABI2, and AtPP2CA genes are highly induced by ABA in guard cells (
      • Leonhardt N.
      • Kwak J.M.
      • Robert N.
      • Waner D.
      • Leonhardt G.
      • Schroeder J.I.
      Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.
      ). The abi1, abi2, atpp2ca, and hab1 mutants exhibit stomatal phenotypes (
      • Leung J.
      • Bouvier-Durand M.
      • Morris P.C.
      • Guerrier D.
      • Chefdor F.
      • Giraudat J.
      Arabidopsis ABA response gene ABI1: features of a calcium-modulated protein phosphatase.
      ,
      • Merlot S.
      • Gosti F.
      • Guerrier D.
      • Vavasseur A.
      • Giraudat J.
      The ABI1 and ABI2 protein phosphatases 2C act in a negative feedback regulatory loop of the abscisic acid signalling pathway.
      ,
      • Leonhardt N.
      • Kwak J.M.
      • Robert N.
      • Waner D.
      • Leonhardt G.
      • Schroeder J.I.
      Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.
      ). In particular, the atpp2ca-1 mutant displays impaired control of the stomatal aperture in epidermal strips in response to ABA (
      • Kuhn J.M.
      • Boisson-Dernier A.
      • Dizon M.B.
      • Maktabi M.H.
      • Schroeder J.I.
      The protein phosphatase AtPP2CA negatively regulates abscisic acid signal transduction in Arabidopsis, and effects of abh1 on AtPP2CA mRNA.
      ). The role of clade A PP2Cs in stomatal closure in response to ABA has been discovered recently (reviewed in Refs.
      • Raghavendra A.S.
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      • Christmann A.
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      ABA perception and signalling.
      ,
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      A brand new START: abscisic acid perception and transduction in the guard cell.
      ). Briefly, the Pyrabactin resistance (PYR)/PYR1-like (PYL)/regulatory component of the ABA receptor (RCAR)-soluble ABA receptors undergo a conformational change upon binding ABA, allowing them to bind and inactivate PP2Cs (
      • Ma Y.
      • Szostkiewicz I.
      • Korte A.
      • Moes D.
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      • Christmann A.
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      Regulators of PP2C phosphatase activity function as abscisic acid sensors.
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      • Cutler S.R.
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      • Finkelstein R.R.
      • Abrams S.R.
      Abscisic acid: emergence of a core signaling network.
      ) and release the SnRK2.6/OST1 kinase. Without ABA, PP2Cs can bind to OST1 and inactivate it (
      • Soon F.F.
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      • Zhou X.E.
      • West G.M.
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      • Cutler S.
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      • Griffin P.R.
      • Melcher K.
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      Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases.
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      • Brandt B.
      • Brodsky D.E.
      • Xue S.
      • Negi J.
      • Iba K.
      • Kangasjärvi J.
      • Ghassemian M.
      • Stephan A.B.
      • Hu H.
      • Schroeder J.I.
      Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action.
      ). Active OST1 mediates anion efflux through the activation of SLAC1 (
      • Lee S.C.
      • Lan W.
      • Buchanan B.B.
      • Luan S.
      A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells.
      ,
      • Geiger D.
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      • Mumm P.
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      • Marten I.
      • Bauer H.
      • Ache P.
      • Matschi S.
      • Liese A.
      • Al-Rasheid K.A.
      • Romeis T.
      • Hedrich R.
      Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.
      ). This signaling pathway is important for guard cell plasma membrane depolarization, which drives K+ efflux and causes stomatal closure. Besides OST1, the calcineurin B-like protein kinase CIPK23 (
      • Maierhofer T.
      • Diekmann M.
      • Offenborn J.N.
      • Lind C.
      • Bauer H.
      • Hashimoto K.S.
      • Al-Rasheid K.A.
      • Luan S.
      • Kudla J.
      • Geiger D.
      • Hedrich R.
      Site- and kinase-specific phosphorylation-mediated activation of SLAC1, a guard cell anion channel stimulated by abscisic acid.
      ) and calcium-dependent protein kinases (CPKs) (
      • Geiger D.
      • Scherzer S.
      • Mumm P.
      • Marten I.
      • Ache P.
      • Matschi S.
      • Liese A.
      • Wellmann C.
      • Al-Rasheid K.A.
      • Grill E.
      • Romeis T.
      • Hedrich R.
      Guard cell anion channel SLAC1 is regulated by CDPK protein kinases with distinct Ca2+ affinities.
      ,
      • Geiger D.
      • Maierhofer T.
      • Al-Rasheid K.A.
      • Scherzer S.
      • Mumm P.
      • Liese A.
      • Ache P.
      • Wellmann C.
      • Marten I.
      • Grill E.
      • Romeis T.
      • Hedrich R.
      Stomatal closure by fast abscisic acid signaling is mediated by the guard cell anion channel SLAH3 and the receptor RCAR1.
      ) also phosphorylate SLAC1 and its homolog SLAH3. This activation is reversed by the PP2Cs. Different mechanisms have been reported for the inhibition of SLAC1 by PP2Cs. For example, ABI1 inhibits SLAC1 activation by CPK6 in Xenopus oocytes and directly dephosphorylates SLAC1 phosphorylated previously by this kinase (
      • Brandt B.
      • Brodsky D.E.
      • Xue S.
      • Negi J.
      • Iba K.
      • Kangasjärvi J.
      • Ghassemian M.
      • Stephan A.B.
      • Hu H.
      • Schroeder J.I.
      Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action.
      ). Conversely, ABI1 does not reverse OST1 phosphorylation of SLAC1 but, instead, interacts physically with OST1, causing an indirect decrease in SLAC1 activity (
      • Geiger D.
      • Scherzer S.
      • Mumm P.
      • Stange A.
      • Marten I.
      • Bauer H.
      • Ache P.
      • Matschi S.
      • Liese A.
      • Al-Rasheid K.A.
      • Romeis T.
      • Hedrich R.
      Activity of guard cell anion channel SLAC1 is controlled by drought-stress signaling kinase-phosphatase pair.
      ). For the SLAC1/AtPP2CA pair, a dual mechanism of inhibition by AtPP2CA has been proposed: an inhibition of the OST1 kinase that activates the SLAC1 channel and a direct interaction with the SLAC1 channel (
      • Lee S.C.
      • Lan W.
      • Buchanan B.B.
      • Luan S.
      A protein kinase-phosphatase pair interacts with an ion channel to regulate ABA signaling in plant guard cells.
      ).
      PP2C phosphatases are not only involved in anion channel regulation but can also modulate plant potassium channels. Shaker channels are voltage-gated, K+-selective, formed by the assembly of four subunits encoded by a family of nine genes in Arabidopsis thaliana. A Shaker subunit comprises a short N-terminal sequence, six transmembrane segments (S1-S6), a pore domain between S5 and S6, and a large intracytoplasmic C-terminal region that comprises about two-thirds of the protein, including a C-linker involved in channel targeting and activity (
      • Nieves-Cordones M.
      • Chavanieu A.
      • Jeanguenin L.
      • Alcon C.
      • Szponarski W.
      • Estaran S.
      • Chérel I.
      • Zimmermann S.
      • Sentenac H.
      • Gaillard I.
      Distinct amino acids in the C-linker domain of the Arabidopsis K+ channel KAT2 determine its subcellular localization and activity at the plasma membrane.
      ), a cyclic nucleotide-binding domain (CNBD), an ankyrin protein-protein interaction domain (absent in KAT1, KAT2, and AtKC1), and a KHA (HA for hydrophobic/acidic) domain (
      • Pilot G.
      • Pratelli R.
      • Gaymard F.
      • Meyer Y.
      • Sentenac H.
      Five-group distribution of the Shaker-like K+ channel family in higher plants.
      ). Depending on the subunits used, inwardly or outwardly rectifying channels can be formed (
      • Dreyer I.
      • Blatt M.R.
      What makes a gate? The ins and outs of Kv-like K+ channels in plants.
      ,
      • Sharma T.
      • Dreyer I.
      • Riedelsberger J.
      The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana.
      ). Most genes encoding subunits involved in inwardly rectifying channel assembly (KAT1, KAT2, AKT1, and AKT2) are expressed in guard cells (
      • Leonhardt N.
      • Kwak J.M.
      • Robert N.
      • Waner D.
      • Leonhardt G.
      • Schroeder J.I.
      Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant.
      ,
      • Szyroki A.
      • Ivashikina N.
      • Dietrich P.
      • Roelfsema M.R.
      • Ache P.
      • Reintanz B.
      • Deeken R.
      • Godde M.
      • Felle H.
      • Steinmeyer R.
      • Palme K.
      • Hedrich R.
      KAT1 is not essential for stomatal opening.
      ,
      • Ivashikina N.
      • Deeken R.
      • Fischer S.
      • Ache P.
      • Hedrich R.
      AKT2/3 subunits render guard cell K+ channels Ca2+ sensitive.
      ), whereas GORK is the only outwardly rectifying channel in this cell type (
      • Hosy E.
      • Vavasseur A.
      • Mouline K.
      • Dreyer I.
      • Gaymard F.
      • Porée F.
      • Boucherez J.
      • Lebaudy A.
      • Bouchez D.
      • Véry A.-A.
      • Simonneau T.
      • Thibaud J.-B.
      • Sentenac H.
      The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration.
      ). Because GORK is activated by membrane depolarization, it is commonly thought that anion efflux initiates K+ efflux through this channel (
      • Sirichandra C.
      • Wasilewska A.
      • Vlad F.
      • Valon C.
      • Leung J.
      The guard cell as a single-cell model towards understanding drought tolerance and abscisic acid action.
      ). In root peripheral cells, AKT1, together with AtKC1, is involved in K+ uptake from the soil. GORK is also located in these cells, although its role remains elusive. GORK may initiate plasma membrane repolarization after depolarization evoked by environmental changes (
      • Sharma T.
      • Dreyer I.
      • Riedelsberger J.
      The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana.
      ,
      • Demidchik V.
      Mechanisms and physiological roles of K+ efflux from root cells.
      ).
      Two Shaker channels, AKT1 and AKT2, have been reported to be regulated by PP2C phosphatases, and a direct interaction has been observed between the phosphatase AtPP2CA and AKT2 (
      • Vranová E.
      • Tähtiharju S.
      • Sriprang R.
      • Willekens H.
      • Heino P.
      • Palva E.T.
      • Inzé D.
      • Van Camp W.
      The AKT3 potassium channel protein interacts with the AtPP2CA protein phosphatase 2C.
      ,
      • Chérel I.
      • Michard E.
      • Platet N.
      • Mouline K.
      • Alcon C.
      • Sentenac H.
      • Thibaud J.-B.
      Physical and functional interaction of the Arabidopsis K+ channel AKT2 and phosphatase AtPP2CA.
      ). Another closely related phosphatase, AIP1/HAI2, binds and regulates AKT1 (
      • Lee S.C.
      • Lan W.Z.
      • Kim B.G.
      • Li L.
      • Cheong Y.-H.
      • Pandey G.K.
      • Lu G.
      • Buchanan B.B.
      • Luan S.
      A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.
      ,
      • Lan W.Z.
      • Lee S.C.
      • Che Y.F.
      • Jiang Y.Q.
      • Luan S.
      Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions.
      ). The AKT1 model predicts that different calcineurin B-like interacting protein kinases (CIPKs) interact with the ankyrin domain to phosphorylate the channel (
      • Lee S.C.
      • Lan W.Z.
      • Kim B.G.
      • Li L.
      • Cheong Y.-H.
      • Pandey G.K.
      • Lu G.
      • Buchanan B.B.
      • Luan S.
      A protein phosphorylation/dephosphorylation network regulates a plant potassium channel.
      ,
      • Lan W.Z.
      • Lee S.C.
      • Che Y.F.
      • Jiang Y.Q.
      • Luan S.
      Mechanistic analysis of AKT1 regulation by the CBL-CIPK-PP2CA interactions.
      ), whereas PP2C phosphatases (including AtPP2CA) inactivate AKT1 by interacting specifically with some of these CIPKs. The direct effect of AIP1 on AKT1 activity was not addressed.
      Inward channel regulation by PP2Cs is now well studied, but nothing is known about outward channel/PP2Cs relationships. Previous studies have shown that GORK and its SKOR (stelar K+ outward rectifier) homolog are sensitive to different signaling factors, such as reactive oxygen species (
      • Demidchik V.
      Mechanisms and physiological roles of K+ efflux from root cells.
      ,
      • Anschütz U.
      • Becker D.
      • Shabala S.
      Going beyond nutrition: regulation of potassium homeostasis as a common denominator of plant adaptative response to environment.
      ,
      • Garcia-Mata C.
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      • Donald N.
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      • Amtmann A.
      • Dreyer I.
      • Blatt M.R.
      A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2.
      ), external K+ (
      • Sharma T.
      • Dreyer I.
      • Riedelsberger J.
      The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana.
      ,
      • Ache P.
      • Becker D.
      • Ivashikina N.
      • Dietrich P.
      • Roelfsema M.R.
      • Hedrich R.
      GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel.
      ,
      • Johansson I.
      • Wulfetange K.
      • Porée F.
      • Michard E.
      • Gajdanowicz P.
      • Lacombe B.
      • Sentenac H.
      • Thibaud J.-B.
      • Mueller-Roeber B.
      • Blatt M.R.
      • Dreyer I.
      External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism.
      ), and internal and external pH (
      • Ache P.
      • Becker D.
      • Ivashikina N.
      • Dietrich P.
      • Roelfsema M.R.
      • Hedrich R.
      GORK, a delayed outward rectifier expressed in guard cells of Arabidopsis thaliana, is a K+-selective, K+-sensing ion channel.
      ,
      • Lacombe B.
      • Pilot G.
      • Gaymard F.
      • Sentenac H.
      • Thibaud J.-B.
      pH control of the plant outwardly-rectifying potassium channel GORK.
      ), and that clustering of GORK channels is correlated with changes in its gating properties (
      • Eisenach C.
      • Papanatsiou M.
      • Hillert E.K.
      • Blatt M.R.
      Clustering of the K+ channel GORK of Arabidopsis parallels its gating by extracellular K+.
      ). Regarding PP2Cs, early physiological studies in guard cells of Nicotiana benthamiana provided evidence that outward K+ fluxes were not driven only by depolarization and that PP2C phosphatases could take part in their control. Overexpression in N. benthamiana of the abi1-1 dominant interfering mutant phosphatase conferring ABA-independent constitutive activity led to a strong and specific decrease of the amplitude of guard cell outward K+ currents independent of the cell membrane potential and not observed with K+ inward and anionic currents (
      • Armstrong F.
      • Leung J.
      • Grabov A.
      • Brearley J.
      • Giraudat J.
      • Blatt M.R.
      Sensitivity to abscisic acid of guard cell K+ channels is suppressed by abil-1, a mutant Arabidopsis gene encoding a putative protein phosphatase.
      ). This indicated that the K+ efflux channel, later identified as GORK, was inhibited by ABI1-1 independent of the membrane potential and ABA signaling pathway.
      In this study, we provide evidence for direct GORK inhibition by AtPP2CA. We also show a strong activation of GORK using phosphomimetic mutations and uncover an unexpected suppression of the activating effect of these mutations by co-expression with AtPP2CA.

      Author Contributions

      C. L., M. B., B. M., A. A. V., and E. M. performed the electrophysiological experiments. E. M. discovered the inhibition of GORK by AtPP2CA. W. S. performed the Western blotting analysis shown in Fig. 7. M. D. helped with the imaging analyses of Fig. 1A. I. G. provided helpful discussions, contributed to the writing of the manuscript, and designed Fig. 5. I. C. designed the study, wrote the paper, finalized the figures, and performed the interaction experiments. All authors analyzed the results and approved the final version of the manuscript.

      Acknowledgments

      We thank Dr. Cornelia Eisenach and Prof. Mike Blatt for the anti-GORK antibody. We also thank Drs. Hervé Sentenac and Nadine Paris for helpful discussions; Dr. Thierry Simonneau for advice regarding plant culture design for transpiration measurements; and Hugues Baudot, Thierry Dessup, and Jose Garcia for plant breeding in the greenhouse. We also thank Dr. Emily Larson for proofreading and critical reading of the manuscript.

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