TY - JOUR A1 - Gonzalez, Wendy A1 - Riedelsberger, Janin A1 - Morales-Navarro, Samuel E. A1 - Caballero, Julio A1 - Alzate-Morales, Jans H. A1 - Gonzalez-Nilo, Fernando D. A1 - Dreyer, Ingo T1 - The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids JF - The biochemical journal N2 - The uptake of potassium ions (K+) accompanied by an acidification of the apoplasm is a prerequisite for stomatal opening. The acidification (approximately 2-2.5 pH units) is perceived by voltage-gated inward potassium channels (K-in) that then can open their pores with lower energy cost. The sensory units for extracellular pH in stomatal K-in channels are proposed to be histidines exposed to the apoplasm. However, in the Arabidopsis thaliana stomatal K-in channel KAT1, mutations in the unique histidine exposed to the solvent (His(267)) do not affect the pH dependency. We demonstrate in the present study that His(267) of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe(266) shifts its pK(a) to undetectable values through a cation-pi interaction. Instead, we show that Glu(240) placed in the extracellular loop between transmembrane segments S5 and S6 is involved in the extracellular acid activation mechanism. Based on structural models we propose that this region may serve as a molecular link between the pH- and the voltage-sensor. Like Glu(240), several other titratable residues could contribute to the pH-sensor of KAT1, interact with each other and even connect such residues far away from the voltage-sensor with the gating machinery of the channel. KW - Arabidopsis thaliana KW - channel protein structure KW - channel protein-proton interaction KW - KAT1 KW - pH regulation KW - potassium chanel Y1 - 2012 U6 - https://doi.org/10.1042/BJ20111498 SN - 0264-6021 VL - 442 IS - 7 SP - 57 EP - 63 PB - Portland Press CY - London ER - TY - JOUR A1 - Garcia-Mata, Carlos A1 - Wang, Jianwen A1 - Gajdanowicz, Pawel A1 - Gonzalez, Wendy A1 - Hills, Adrian A1 - Donald, Naomi A1 - Riedelsberger, Janin A1 - Amtmann, Anna A1 - Dreyer, Ingo A1 - Blatt, Michael R. T1 - A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2 N2 - 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. Y1 - 2010 UR - http://www.jbc.org/ U6 - https://doi.org/10.1074/jbc.M110.141176 SN - 0021-9258 ER - TY - JOUR A1 - Lefoulon, Cecile A1 - Karnik, Rucha A1 - Honsbein, Annegret A1 - Gutla, Paul Vijay A1 - Grefen, Christopher A1 - Riedelsberger, Janin A1 - Poblete, Tomas A1 - Dreyer, Ingo A1 - Gonzalez, Wendy A1 - Blatt, Michael R. T1 - Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor JF - Plant physiology : an international journal devoted to physiology, biochemistry, cellular and molecular biology, biophysics and environmental biology of plants N2 - 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. Y1 - 2014 U6 - https://doi.org/10.1104/pp.114.244319 SN - 0032-0889 SN - 1532-2548 VL - 166 IS - 2 SP - 960 EP - U776 PB - American Society of Plant Physiologists CY - Rockville ER - TY - JOUR A1 - Riedelsberger, Janin A1 - Dreyer, Ingo A1 - Gonzalez, Wendy T1 - Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History JF - PLoS one N2 - Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom. Y1 - 2015 U6 - https://doi.org/10.1371/journal.pone.0137600 SN - 1932-6203 VL - 10 IS - 9 PB - PLoS CY - San Fransisco ER - TY - GEN A1 - Riedelsberger, Janin A1 - Dreyer, Ingo A1 - Gonzalez, Wendy T1 - Outward rectification of voltage-gated K+ channels evolved at least twice in life history T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Voltage-gated potassium (K+) channels are present in all living systems. Despite high structural similarities in the transmembrane domains (TMD), this K+ channel type segregates into at least two main functional categories-hyperpolarization-activated, inward-rectifying (Kin) and depolarization-activated, outward-rectifying (Kout) channels. Voltage-gated K+ channels sense the membrane voltage via a voltage-sensing domain that is connected to the conduction pathway of the channel. It has been shown that the voltage-sensing mechanism is the same in Kin and Kout channels, but its performance results in opposite pore conformations. It is not known how the different coupling of voltage-sensor and pore is implemented. Here, we studied sequence and structural data of voltage-gated K+ channels from animals and plants with emphasis on the property of opposite rectification. We identified structural hotspots that alone allow already the distinction between Kin and Kout channels. Among them is a loop between TMD S5 and the pore that is very short in animal Kout, longer in plant and animal Kin and the longest in plant Kout channels. In combination with further structural and phylogenetic analyses this finding suggests that outward-rectification evolved twice and independently in the animal and plant kingdom. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 521 KW - multiple sequence alignment KW - potassium channel KW - Arabidopsis thaliana KW - inward rectification KW - pacemaker channels KW - S4-S5 linker KW - sensor KW - expression KW - mechanism KW - activation Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-409594 SN - 1866-8372 IS - 521 ER -