TY - JOUR A1 - Dreyer, Ingo A1 - Poree, Fabien A1 - Schneider, A. A1 - Mittelstadt, J. A1 - Bertl, Adam A1 - Sentenac, H. A1 - Thibaud, Jean-Baptiste A1 - Müller-Röber, Bernd T1 - Assembly of plant Shaker-like K-out channels requires two distinct sites of the channel alpha-subunit N2 - SKOR and GORK are outward-rectifying plant potassium channels from Arabidopsis thaliana. They belong to the Shaker superfamily of voltage-dependent K+ channels. Channels of this class are composed of four alpha-subunits and subunit assembly is a prerequisite for channel function. In this study the assembly mechanism of SKOR was investigated using the yeast two-hybrid system and functional assays in Xenopus oocytes and in yeast. We demonstrate that SKOR and GORK physically interact and assemble into heteromeric K-out channels. Deletion mutants and chimeric proteins generated from SKOR and the K-in channel alpha-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains thatchannel a-subunit KAT1 revealed that the cytoplasmic C-terminus of SKOR determines channel assembly. Two domains that are crucial for channel assembly were identified: i), a proximal interacting region comprising a putative cyclic nucleotide-binding domain together with 33 amino acids just upstream of this domain, and ii), a distal interacting region showing some resemblance to the K-T domain of KAT1. Both regions contributed differently to channel assembly. Whereas the proximal interacting region was found to be active on its own, the distal interacting region required an intact proximal interacting region to be active. K-out alpha-subunits did not assemble with K-in alpha-subunits because of the absence of interaction between their assembly sites Y1 - 2004 SN - 0006-3495 ER - TY - JOUR A1 - Poree, Fabien A1 - Wulfetange, K. A1 - Naso, A. A1 - Carpaneto, Armando A1 - Roller, A. A1 - Natura, G. A1 - Bertl, Adam A1 - Sentenac, H. A1 - Thibaud, Jean-Baptiste A1 - Dreyer, Ingo T1 - Plant K-in and K-out channels : Approaching the trait of opposite rectification by analyzing more than 250 KAT1- SKOR chimeras N2 - Members of the Shaker-like plant K+ channel family share a common structure, but are highly diverse in their function: they behave as either hyperpolarization-activated inward-rectifying (K-in) channels, or leak-like (K-weak) channels, or depolarization-activated outward-rectifying (K-out) channels. Here we created 256 chimeras between the K-in channel KAT1 and the K-out channel SKOR. The chimeras were screened in a potassium-uptake deficient yeast strain to identify those, which mediate potassium inward currents, i.e., which are functionally equivalent to KAT1. This strategy allowed Lis to identify three chimeras which differ from KAT1 in three parts of the polypeptide: the cytosolic N- terminus, the cytosolic C-terminus, and the putative voltage-sensor S4. Additionally, mutations in the K-out Channel SKOR were generated in order to localize molecular entities underlying its depolarization activation. The triple mutant SKOR-D312N-M313L-1314G, carrying amino-acid changes in the S6 segment, was identified as a channel which did not display any rectification in the tested voltage-range. (C) 2005 Elsevier Inc. All rights reserved Y1 - 2005 SN - 0006-291X ER - TY - JOUR A1 - Michard, Erwan A1 - Lacombe, Benoît A1 - Poree, Fabien A1 - Müller-Röber, Bernd A1 - Sentenac, Hervé A1 - Thibaud, Jean-Baptiste A1 - Dreyer, Ingo T1 - A unique voltage sensor sensitizes the potassium channel AKT2 to phosphoregulation N2 - Among all voltage-gated K+ channels from the model plant Arabidopsis thaliana, the weakly rectifying K+ channel (K-weak channel) AKT2 displays unique gating properties. AKT2 is exceptionally regulated by phosphorylation: when nonphosphorylated AKT2 behaves as an inward-rectifying potassium channel; phosphorylation of AKT2 abolishes inward rectification by shifting its activation threshold far positive (>200 mV) so that it closes only at voltages positive of + 100 mV. In its phosphorylated form, AKT2 is thus locked in the open state in the entire physiological voltage range. To understand the molecular grounds of this unique gating behavior, we generated chimeras between AKT2 and the conventional inward-rectifying channel KAT1. The transfer of the pore from KAT1 to AKT2 altered the permeation properties of the channel. However, the gating properties were unaffected, suggesting that the pore region of AKT2 is not responsible for the unique K-weak gating. Instead, a lysine residue in S4, highly conserved among all K-weak channels but absent from other plant K+ channels, was pinpointed in a site-directed mutagenesis approach. Substitution of the lysine by serine or aspartate abolished the "open-lock" characteristic and converted AKT2 into an inward- rectifying channel. Interestingly, phosphoregulation of the mutant AKT2-K197S appeared to be similar to that of the K-in channel KAT1: as suggested by mimicking the phosphorylated and dephosphorylated states, phosphorylation induced a shift of the activation threshold of AKT2-K197S by about +50 mV. We conclude that the lysine residue K197 sensitizes AKT2 to phosphoregulation. The phosphorylation-induced reduction of the activation energy in AKT2 is similar to 6 kT larger than in the K197S mutant. It is discussed that this hypersensitive response of AKT2 to phosphorylation equips a cell with the versatility to establish a potassium gradient and to make efficient use of it Y1 - 2005 ER - TY - JOUR A1 - Riano-Pachon, Diego Mauricio A1 - Dreyer, Ingo A1 - Müller-Röber, Bernd T1 - Orphan transcripts in Arabidopsis thaliana : identification of several hundred previously unrecognized genes N2 - Expressed sequence tags (ESTs) represent a huge resource for the discovery of previously unknown genetic information and functional genome assignment. In this study we screened a collection of 178 292 ESTs from Arabidopsis thaliana by testing them against previously annotated genes of the Arabidopsis genome. We identified several hundreds of new transcripts that match the Arabidopsis genome at so far unassigned loci. The transcriptional activity of these loci was independently confirmed by comparison with the Salk Whole Genome Array Data. To a large extent, the newly identified transcriptionally active genomic regions do not encode 'classic' proteins, but instead generate non-coding RNAs and/or small peptide-coding RNAs of presently unknown biological function. More than 560 transcripts identified in this study are not represented by the Affymetrix GeneChip arrays currently widely used for expression profiling in A. thaliana. Our data strongly support the hypothesis that numerous previously unknown genes exist in the Arabidopsis genome Y1 - 2005 SN - 0960-7412 ER - TY - JOUR A1 - Sato, A A1 - Gambale, Franco A1 - Dreyer, Ingo A1 - Uozumi, N T1 - Posttranslational inodification affects K+ current of plant K+ channel Y1 - 2006 ER - TY - JOUR A1 - Wood, C. C. A1 - Poree, Fabien A1 - Dreyer, Ingo A1 - Koehler, G. J. A1 - Udvardi, M. K. T1 - Mechanisms of ammonium transport, accumulation, and retention in ooyctes and yeast cells expressing Arabidopsis AtAMT1; 1 N2 - Ammonium is a primary source of N for plants, so knowing how it is transported, stored, and assimilated in plant cells is important for rational approaches to optimise N-use in agriculture. Electrophysiological studies of Arabidopsis AtAMT1;1 expressed in oocytes revealed passive, Delta psi-driven transport of NH4+ through this protein. Expression of AtAMT1;1 in a novel yeast mutant defective in endogenous ammonium transport and vacuolar acidification supported the above mechanism for AtAMT1;1 and revealed a central role for acid vacuoles in storage and retention of ammonia in cells. These results highlight the mechanistic differences between plant AMT proteins and related transporters in bacteria and animal cells, and suggest novel strategies to enhance nitrogen use efficiency in agriculture. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved Y1 - 2006 UR - http://www.sciencedirect.com/science/article/pii/S0014579306007332 U6 - https://doi.org/10.1016/j.febslet.2006.06.026 ER - TY - JOUR A1 - Johansson, Ingela A1 - Wulfetange, Klaas A1 - Poree, Fabien A1 - Michard, Erwan A1 - Gajdanowicz, Pawel A1 - Lacombe, Benoit A1 - Sentenac, Herve A1 - Thibaud, Jean-Baptiste A1 - Müller-Röber, Bernd A1 - Blatt, Michael R. A1 - Dreyer, Ingo T1 - External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism N2 - Plant outward-rectifying K+ channels mediate K+ efflux from guard cells during stomatal closure and from root cells into the xylem for root-shoot allocation of potassium (K). Intriguingly, the gating of these channels depends on the extracellular K+ concentration, although the ions carrying the current are derived from inside the cell. This K+ dependence confers a sensitivity to the extracellular K+ concentration ([K+]) that ensures that the channels mediate K+ efflux only, regardless of the [K+] prevailing outside. We investigated the mechanism of K+-dependent gating of the K+ channel SKOR of Arabidopsis by site-directed mutagenesis. Mutations affecting the intrinsic K+ dependence of gating were found to cluster in the pore and within the sixth transmembrane helix (S6), identifying an 'S6 gating domain' deep within the membrane. Mapping the SKOR sequence to the crystal structure of the voltage-dependent K+ channel KvAP from Aeropyrum pernix suggested interaction between the S6 gating domain and the base of the pore helix, a prediction supported by mutations at this site. These results offer a unique insight into the molecular basis for a physiologically important K+-sensory process in plants Y1 - 2006 UR - http://www3.interscience.wiley.com/cgi-bin/issn?DESCRIPTOR=PRINTISSN&VALUE=0960-7412 U6 - https://doi.org/10.1111/j.1365-313X.2006.02690.X SN - 0960-7412 ER - TY - JOUR A1 - Dreyer, Ingo A1 - Gajdanowicz, Pawel T1 - Regulation of the gating mode of the Arabidopsis K+ channel AKT2 is important for adaptation to abiotic stress Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/10956433 U6 - https://doi.org/10.1016/j.cbpa.2009.04.426 SN - 1095-6433 ER - TY - JOUR A1 - Dreyer, Ingo A1 - Blatt, Michael R. T1 - What makes a gate? : the ins and outs of Kv-like K+ channels in plants N2 - Gating of K+ and other ion channels is 'hard-wired' within the channel protein. So it remains a puzzle how closely related channels in plants can show an unusually diverse range of biophysical properties. Gating of these channels lies at the heart of K+ mineral nutrition, signalling, abiotic and biotic stress responses in plants. Thus, our knowledge of the molecular mechanics underpinning K+ channel gating will be important for rational engineering of related traits in agricultural crops. Several key studies have added significantly to our understanding of channel gating in plants and have challenged current thinking about analogous processes found in animal K+ channels. Such studies highlight how much of K+ channel gating remains to be explored in plants. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/13601385 U6 - https://doi.org/10.1016/j.tplants.2009.04.001 SN - 1360-1385 ER - TY - JOUR A1 - Naso, Alessia A1 - Dreyer, Ingo A1 - Pedemonte, Laura A1 - Testa, Ilaria A1 - Gomez-Porras, Judith Lucia A1 - Usai, Cesare A1 - Müller-Röber, Bernd A1 - Diaspro, Alberto A1 - Gambale, Franco A1 - Picco, Cristiana T1 - The role of the C-terminus for functional heteromerization of the plant channel KDC1 N2 - Voltage-gated potassium channels are formed by the assembly of four identical (homotetramer) or different (heterotetramer) subunits. Tetramerization of plant potassium channels involves the C-terminus of the protein. We investigated the role of the C-terminus of KDC1, a Shaker-like inward-rectifying K+ channel that does not form functional homomeric channels, but participates in the formation of heteromeric complexes with other potassium alpha- subunits when expressed in Xenopus oocytes. The interaction of KDC1 with KAT1 was investigated using the yeast two- hybrid system, fluorescence and electrophysiological studies. We found that the KDC1-EGFP fusion protein is not targeted to the plasma membrane of Xenopus oocytes unless it is coexpressed with KAT1. Deletion mutants revealed that the KDC1 C- terminus is involved in heteromerization. Two domains of the C-terminus, the region downstream the putative cyclic nucleotide binding domain and the distal part of the C-terminus called K-HA domain, contributed to a different extent to channel assembly. Whereas the first interacting region of the C-terminus was necessary for channel heteromerization, the removal of the distal KHA domain decreased but did not abolish the formation of heteromeric complexes. Similar results were obtained when coexpressing KDC1 with the KAT1-homolog KDC2 from carrots, thus indicating the physiological significance of the KAT1/KDC1 characterization. Electrophysiological experiments showed furthermore that the heteromerization capacity of KDC1 was negatively influenced by the presence of the enhanced green fluorescence protein fusion. Y1 - 2009 UR - http://www.sciencedirect.com/science/journal/00063495 U6 - https://doi.org/10.1016/j.bpj.2009.02.055 SN - 0006-3495 ER -