@article{LefoulonKarnikHonsbeinetal.2014, author = {Lefoulon, Cecile and Karnik, Rucha and Honsbein, Annegret and Gutla, Paul Vijay and Grefen, Christopher and Riedelsberger, Janin and Poblete, Tomas and Dreyer, Ingo and Gonzalez, Wendy and Blatt, Michael R.}, title = {Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor}, series = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, volume = {166}, journal = {Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants}, number = {2}, publisher = {American Society of Plant Physiologists}, address = {Rockville}, issn = {0032-0889}, doi = {10.1104/pp.114.244319}, pages = {960 -- U776}, year = {2014}, abstract = {The Kv-like (potassium voltage-dependent) K+ channels at the plasma membrane, including the inward-rectifying KAT1 K+ channel of Arabidopsis (Arabidopsis thaliana), are important targets for manipulating K+ homeostasis in plants. Gating modification, especially, has been identified as a promising means by which to engineer plants with improved characteristics in mineral and water use. Understanding plant K+ channel gating poses several challenges, despite many similarities to that of mammalian Kv and Shaker channel models. We have used site-directed mutagenesis to explore residues that are thought to form two electrostatic countercharge centers on either side of a conserved phenylalanine (Phe) residue within the S2 and S3 alpha-helices of the voltage sensor domain (VSD) of Kv channels. Consistent with molecular dynamic simulations of KAT1, we show that the voltage dependence of the channel gate is highly sensitive to manipulations affecting these residues. Mutations of the central Phe residue favored the closed KAT1 channel, whereas mutations affecting the countercharge centers favored the open channel. Modeling of the macroscopic current kinetics also highlighted a substantial difference between the two sets of mutations. We interpret these findings in the context of the effects on hydration of amino acid residues within the VSD and with an inherent bias of the VSD, when hydrated around a central Phe residue, to the closed state of the channel.}, language = {en} } @article{JohanssonWulfetangePoreeetal.2006, author = {Johansson, Ingela and Wulfetange, Klaas and Poree, Fabien and Michard, Erwan and Gajdanowicz, Pawel and Lacombe, Benoit and Sentenac, Herve and Thibaud, Jean-Baptiste and M{\"u}ller-R{\"o}ber, Bernd and Blatt, Michael R. and Dreyer, Ingo}, title = {External K+ modulates the activity of the Arabidopsis potassium channel SKOR via an unusual mechanism}, issn = {0960-7412}, doi = {10.1111/j.1365-313X.2006.02690.X}, year = {2006}, abstract = {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}, language = {en} } @article{GarciaMataWangGajdanowiczetal.2010, author = {Garcia-Mata, Carlos and Wang, Jianwen and Gajdanowicz, Pawel and Gonzalez, Wendy and Hills, Adrian and Donald, Naomi and Riedelsberger, Janin and Amtmann, Anna and Dreyer, Ingo and Blatt, Michael R.}, title = {A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2}, issn = {0021-9258}, doi = {10.1074/jbc.M110.141176}, year = {2010}, abstract = {Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca2+ and K+ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K+ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H2O2. We show that H2O2 rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H2O2 and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H2O2-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys(168) located within the S3 alpha-helix of the voltage sensor complex, to be essential for sensitivity to H2O2. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys168 as a critical target for H2O2, and implicate ROS-mediated control of the K+ channel in regulating mineral nutrient partitioning within the plant.}, language = {en} } @article{GajdanowiczGarciaMataGonzalezetal.2009, author = {Gajdanowicz, Pawel and Garcia-Mata, Carlos and Gonzalez, Wendy and Morales-Navarro, Samuel El{\"i}as and Sharma, Tripti and Gonzalez-Nilo, Fernando Danilo and Gutowicz, Jan and M{\"u}ller-R{\"o}ber, Bernd and Blatt, Michael R. and Dreyer, Ingo}, title = {Distinct roles of the last transmembrane domain in controlling Arabidopsis K+ channel activity}, issn = {0028-646X}, doi = {10.1111/j.1469-8137.2008.02749.x}, year = {2009}, abstract = {The family of voltage-gated potassium channels in plants presumably evolved from a common ancestor and includes both inward-rectifying (K-in) channels that allow plant cells to accumulate K+ and outward-rectifying (K-out) channels that mediate K+ efflux. Despite their close structural similarities, the activity of Kin channels is largely independent of K+ and depends only on the transmembrane voltage, whereas that of K-out channels responds to the membrane voltage and the prevailing extracellular K+ concentration. Gating of potassium channels is achieved by structural rearrangements within the last transmembrane domain (S6). Here we investigated the functional equivalence of the S6 helices of the Kin channel KAT1 and the K-out channel SKOR by domain-swapping and site-directed mutagenesis. Channel mutants and chimeras were analyzed after expression in Xenopus oocytes. We identified two discrete regions that influence gating differently in both channels, demonstrating a lack of functional complementarity between KAT1 and SKOR. Our findings are supported by molecular models of KAT1 and SKOR in the open and closed states. The role of the S6 segment in gating evolved differently during specialization of the two channel subclasses, posing an obstacle for the transfer of the K+-sensor from K-out to K-in channels.}, language = {en} } @article{DreyerBlatt2009, author = {Dreyer, Ingo and Blatt, Michael R.}, title = {What makes a gate? : the ins and outs of Kv-like K+ channels in plants}, issn = {1360-1385}, doi = {10.1016/j.tplants.2009.04.001}, year = {2009}, abstract = {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.}, language = {en} }