Janin Riedelsberger

A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2

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, Cys168 located within the S3 α-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.

The role of K+ channels in uptake and redistribution of potassium in the model plant Arabidopsis thaliana

Potassium (K+) is inevitable for plant growth and development. It plays a crucial role in the regulation of enzyme activities, in adjusting the electrical membrane potential and the cellular turgor, in regulating cellular homeostasis and in the stabilization of protein synthesis. Uptake of K+ from the soil and its transport to growing organs is essential for a healthy plant development. Uptake and allocation of K+ are performed by K+ channels and transporters belonging to different protein families. In this review we summarize the knowledge on the versatile physiological roles of plant K+ channels and their behavior under stress conditions in the model plant Arabidopsis thaliana.

Distributed structures underlie gating differences between the Kin channel KAT1 and the Kout channel SKOR.

The family of voltage-gated (Shaker-like) potassium channels in plants 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, K(in) and K(out) channels differ in their gating sensitivity towards voltage and the extracellular K(+) concentration. We have carried out a systematic program of domain swapping between the K(out) channel SKOR and the K(in) channel KAT1 to examine the impacts on gating of the pore regions, the S4, S5, and the S6 helices. We found that, in particular, the N-terminal part of the S5 played a critical role in KAT1 and SKOR gating. Our findings were supported by molecular dynamics of KAT1 and SKOR homology models. In silico analysis revealed that during channel opening and closing, displacement of certain residues, especially in the S5 and S6 segments, is more pronounced in KAT1 than in SKOR. From our analysis of the S4-S6 region, we conclude that gating (and K(+)-sensing in SKOR) depend on a number of structural elements that are dispersed over this approximately 145-residue sequence and that these place additional constraints on configurational rearrangement of the channels during gating.

Voltage-sensor transitions of the inward-rectifying K+ channel KAT1 indicate a latching mechanism biased by hydration within the voltage sensor

Manipulating the electrostatic charge network that stabilizes the voltage sensor of the KAT1 K+ channel displaces channel gating across more than 140 mV within the physiological voltage range. 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 α-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.

The pH sensor of the plant K+-uptake channel KAT1 is built from a sensory cloud rather than from single key amino acids.

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 (His267) do not affect the pH dependency. We demonstrate in the present study that His267 of the KAT1 channel cannot sense pH changes since the neighbouring residue Phe266 shifts its pKa to undetectable values through a cation-π interaction. Instead, we show that Glu240 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 Glu240, 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.

The potassium battery: a mobile energy source for transport processes in plant vascular tissues

Contents 1049 I. 1049 II. 1050 III. 1050 IV. 1050 V. 1051 VI. 1051 VII. 1052 VIII. 1052 1053 References 1053 SUMMARY: Plant roots absorb potassium ions from the soil and transport them in the xylem via the transpiration stream to the shoots. There, in source tissues where sufficient chemical energy (ATP) is available, K+ is loaded into the phloem and then transported with the phloem stream to other parts of the plant; in part, transport is also back to the roots. This, at first sight, futile cycling of K+ has been uncovered to be part of a sophisticated mechanism that (1) enables the shoot to communicate its nutrient demand to the root, (2) contributes to the K+ nutrition of transport phloem tissues and (3) transports energy stored in the K+ gradient between phloem cytosol and the apoplast. This potassium battery can be tapped by opening AKT2-like potassium channels and then enables the ATP-independent energization of other transport processes, such as the reloading of sucrose. Insights into these mechanisms have only been possible by combining wet-lab and dry-lab experiments by means of computational cell biology modeling and simulations.

K2P channels in plants and animals

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H+ and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

Outward Rectification of Voltage-Gated K+ Channels Evolved at Least Twice in Life History

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.

Editorial: Roots—The Hidden Provider

Most plant roots are found hidden underground. They can form immense root systems with lengths of several kilometers featuring an architecture with up to millions of branch roots. Their versatile functions range from anchoring plants in soil via storing photosynthetic products to the vital uptake of water and nutrients. Plants build the basis of a food chain that ends with more than seven billion people who have to be fed every day. Therefore, it is vital to secure and enhance crop production, especially in terms of prevailing agricultural threats and climate change. The challengemust be to optimize plant’s nutrient acquisition and stress tolerance. Understanding plant nutrition, homeostasis, and stress responses is a first step to directed and efficient manipulations and adjustments to counterbalance stress and deficiency symptoms. This knowledge must be used to optimize the use of fertilizer and water to achieve well-balanced nutrition acquisition as well as the use of pesticides to treat biotic stress. Such optimization is desirable as the constant use of fertilizer, irrigation, and pesticides is associated with negative long-term effects. Knowledge about nutrient uptake and signaling as well as behavior in stress situations will be valuable if we are to design adequate treatments to prepare plants for environmental challenges.