The essential mineral nutrient potassium (K + ) is the most important inorganic cation for plants and is recognized as a limiting factor for crop yield and quality. Nonetheless, it is only partially understood how K + contributes to plant productivity. K + is used as a major active solute to maintain turgor and to drive irreversible and reversible changes in cell volume. K + also plays an important role in numerous metabolic processes, for example, by serving as an essential cofactor of enzymes. Here, we provide evidence for an additional, previously unrecognized role of K + in plant growth. By combining diverse experimental approaches with computational cell simulation, we show that K + circulating in the phloem serves as a decentralized energy storage that can be used to overcome local energy limitations. Posttranslational modification of the phloem-expressed Arabidopsis K + channel AKT2 taps this "potassium battery," which then efficiently assists the plasma membrane H + -ATPase in energizing the transmembrane phloem (re) loading processes.channel gating | energy limiting condition | phloem reloading | posttranslational regulation | potassium channel
Potassium (K+) channels have been evolutionarily tuned for activation by diverse biological stimuli, and pharmacological activation is thought to target these specific gating mechanisms. Here we report a class of negatively charged activators (NCAs) that bypass the specific mechanisms but act as master keys to open K+channels gated at their selectivity filter (SF), including many two-pore domain K+(K2P) channels, voltage-gated hERG (human ether-à-go-go–related gene) channels and calcium (Ca2+)–activated big-conductance potassium (BK)–type channels. Functional analysis, x-ray crystallography, and molecular dynamics simulations revealed that the NCAs bind to similar sites below the SF, increase pore and SF K+occupancy, and open the filter gate. These results uncover an unrecognized polypharmacology among K+channel activators and highlight a filter gating machinery that is conserved across different families of K+channels with implications for rational drug design.
Potassium channels share a common selectivity filter that determines the conduction characteristics of the pore. Diversity in K ؉ channels is given by how they are gated open. TASK-2, TALK-1, and TALK-2 are two-pore region (2P) KCNK K ؉ channels gated open by extracellular alkalinization. We have explored the mechanism for this alkalinization-dependent gating using molecular simulation and site-directed mutagenesis followed by functional assay. We show that the side chain of a single arginine residue (R224) near the pore senses pH in TASK-2 with an unusual pK a of 8.0, a shift likely due to its hydrophobic environment. R224 would block the channel through an electrostatic effect on the pore, a situation relieved by its deprotonation by alkalinization. A lysine residue in TALK-2 fulfills the same role but with a largely unchanged pK a, which correlates with an environment that stabilizes its positive charge. In addition to suggesting unified alkaline pH-gating mechanisms within the TALK subfamily of channels, our results illustrate in a physiological context the principle that hydrophobic environment can drastically modulate the pK a of charged amino acids within a protein.KCNK channels ͉ molecular simulation ͉ TALK-2 ͉ TASK-2 A ll K ϩ channels contain a highly conserved sequence, the P domain, which forms the selectivity filter and generally six transmembrane ␣-helices. The K ϩ channel pore is formed by four identical subunits, each comprising a P-domain and two of the six transmembrane ␣-helices encircling the ion conduction pathway with a 4-fold symmetry. Structures attached to the pore-forming domains are able to transduce signals, such as changes in transmembrane voltage and intra-or extracellular messages, into gating of the pore (1, 2). Potassium channels of the KCNK superfamily (3, 4) are remarkable in that they possess two P-domains and four ␣-helices in each subunit, form dimers, and are mostly open (''leak channels'') at resting potential. Potassium-selective leaks are fundamental to the function of various cells including nerve, muscle, and epithelia. There are 16 mammalian members to the KCNK family and their gating is variously regulated by free fatty acids, membrane tension, G protein-generated signaling, and extracellular pH. Among KCNK channels gated by extracellular pH, TASK-1 and TASK-3 form a subfamily (TASK) of channels blocked by extracellular protons (5-8). A second subfamily (TALK) of KCNK channels comprises TASK-2, TALK-1, and TALK-2 ¶ , all activated by extracellular alkalinization. TASK-2 participates in ion fluxes necessary for cell volume regulation (10, 11), and its physiological and possible pathological importance has also been highlighted by studies in a TASK-2 knockout mouse (12) that revealed a metabolic acidosis and hypotension caused by renal loss of HCO K ϩ channels of the TASK subfamily are blocked by protons by titration of a histidine (N in TALK channels) residue in the first P domain (6-8), making them responsive to pH in the physiological range. The pH-sensing mechanism ...
Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They Reactive oxygen species (ROS) 3 are major signals in virtually every aspect of eukaryotic cell biology (1, 2). In animals, they are important regulators of cell division and muscle contraction among others (3), and in plants they are essential for development (4) and stress signaling, including drought (5, 6) and defense against pathogens (7). Within proteins, ROS target a small subset of amino acids, notably cysteine (Cys) residues, chemically modifying these amino acids and thereby altering protein structure and function (8). Nonetheless, the targeted residues and associated motifs are often poorly defined, the effects wide ranging and protein-specific, thus confounding molecular analyses. Indeed, among the few well documented examples, ROS modifications in mammalian ryanodine-receptor and BK channels have different consequences depending on the positions of the residues targeted (3, 9).For development and signaling in plants, the activities of membrane ion channels are essential, often including their regulation by ROS. For example, ROS affect non-selective cation channels during Fucus development (10); they regulate Ca 2ϩ channels and Ca 2ϩ -based signaling (5) as well as voltage-sensitive K ϩ channels of guard cells (11) that are important for stomatal movement; they contribute in responses to drought and pathogen defense (12); and they have been implicated in targeting K ϩ efflux during programmed cell death (13). However, until now virtually nothing has been known of the molecular targets for ROS in plants, nor has a site of action been identified with an ion channel protein.SKOR is one of two K ϩ channels found in Arabidopsis thaliana that rectify strongly outward, thereby mediating K ϩ efflux from the cell. It is expressed within the xylem parenchyma of the root where it facilitates K ϩ loading into the xylem (14), thereby contributing directly to K ϩ homeostasis and indirectly, through charge balance, to the transport of other solutes throughout the plant (12,15). Like other members of the Kvlike channel superfamily (16), the functional channel assembles from four monomers with each SKOR monomer incorporating six transmembrane ␣-helices. The first four ␣-helices form a positively charged voltage-sensor complex or "paddle" that moves within the membrane in response to voltage and couples this movement to the channel gate. The fifth and sixth ␣-helices line the aqueous pore through the membrane and assemble in a diaphragm or "gate" at the inner membrane surface, which opens/closes to regulate ion flux through the channel. Here we report that K ϩ current through the heterologously expressed SKOR is modulated by H 2 O 2 , thereby identifying the K ϩ channel as a potential target for ROS, and we show that a single Cys within the voltage sensor complex is essential for its ROS sen-
Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.
Atrial fibrillation and obstructive sleep apnea are responsible for significant morbidity and mortality in the industrialized world. There is a high medical need for novel drugs against both diseases, and here, Kv1.5 channels have emerged as promising drug targets. In humans, TASK-1 has an atrium-specific expression and TASK-1 is also abundantly expressed in the hypoglossal motor nucleus. We asked whether known Kv1.5 channel blockers, effective against atrial fibrillation and/or obstructive sleep apnea, modulate TASK-1 channels. Therefore, we tested Kv1.5 blockers with different chemical structures for their TASK-1 affinity, utilizing two-electrode voltage clamp (TEVC) recordings in Xenopus oocytes. Despite the low structural conservation of Kv1.5 and TASK-1 channels, we found all Kv1.5 blockers analyzed to be even more effective on TASK-1 than on Kv1.5. For instance, the half-maximal inhibitory concentration (IC50) values of AVE0118 and AVE1231 (A293) were 10- and 43-fold lower on TASK-1. Also for MSD-D, ICAGEN-4, S20951 (A1899), and S9947, the IC50 values were 1.4- to 70-fold lower than for Kv1.5. To describe this phenomenon on a molecular level, we used in silico models and identified unexpected structural similarities between the two drug binding sites. Kv1.5 blockers, like AVE0118 and AVE1231, which are promising drugs against atrial fibrillation or obstructive sleep apnea, are in fact potent TASK-1 blockers. Accordingly, block of TASK-1 channels by these compounds might contribute to the clinical effectiveness of these drugs. The higher affinity of these blockers for TASK-1 channels suggests that TASK-1 might be an unrecognized molecular target of Kv1.5 blockers effective in atrial fibrillation or obstructive sleep apnea.
In a patient with right ventricular outflow tract (RVOT) tachycardia, we identified a heterozygous point mutation in the selectivity filter of the stretch‐activated K2P potassium channel TREK‐1 (KCNK2 or K2P2.1). This mutation introduces abnormal sodium permeability to TREK‐1. In addition, mutant channels exhibit a hypersensitivity to stretch‐activation, suggesting that the selectivity filter is directly involved in stretch‐induced activation and desensitization. Increased sodium permeability and stretch‐sensitivity of mutant TREK‐1 channels may trigger arrhythmias in areas of the heart with high physical strain such as the RVOT. We present a pharmacological strategy to rescue the selectivity defect of the TREK‐1 pore. Our findings provide important insights for future studies of K2P channel stretch‐activation and the role of TREK‐1 in mechano‐electrical feedback in the heart.
The LEA (late embryogenesis abundant) proteins COR15A and COR15B from Arabidopsis thaliana are intrinsically disordered under fully hydrated conditions, but obtain α-helical structure during dehydration, which is reversible upon rehydration. To understand this unusual structural transition, both proteins were investigated by circular dichroism (CD) and molecular dynamics (MD) approaches. MD simulations showed unfolding of the proteins in water, in agreement with CD data obtained with both HIS-tagged and untagged recombinant proteins. Mainly intramolecular hydrogen bonds (H-bonds) formed by the protein backbone were replaced by H-bonds with water molecules. As COR15 proteins function in vivo as protectants in leaves partially dehydrated by freezing, unfolding was further assessed under crowded conditions. Glycerol reduced (40%) or prevented (100%) unfolding during MD simulations, in agreement with CD spectroscopy results. H-bonding analysis indicated that preferential exclusion of glycerol from the protein backbone increased stability of the folded state.
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